Methods for the treatment of HER2 amplified cancer

ABSTRACT

Described herein are methods and compositions for treating HER2-amplified cancer. The methods include administering to an individual in need thereof ibrutinib.

RELATED APPLICATION

The present application claims the benefit of priority from U.S.Provisional Patent Application Nos. 61/865,059, filed Aug. 12, 2013, and61/969,003, filed Mar. 21, 2014, each of which is herein incorporated byreference in its entirety.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions for use in treating HER2 amplified breast cancer in apatient, wherein the pharmaceutical composition comprises(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

In some embodiments, a dose of ibrutinib is from about 100 mg/day up to,and including, about 2000 mg/day. In some embodiments, a dose ofibrutinib is from about 140 mg/day up to, and including, about 840mg/day. In some embodiments, a dose of ibrutinib is from about 420mg/day up to, and including, about 840 mg/day. In some embodiments, adose of ibrutinib is about 140 mg/day. In some embodiments, a dose ofibrutinib is about 280 mg/day. In some embodiments, a dose of ibrutinibis about 420 mg/day. In some embodiments, a dose of ibrutinib is about560 mg/day. In some embodiments, a dose of ibrutinib is about 700mg/day. In some embodiments, a dose of ibrutinib is about 840 mg/day. Insome embodiments, a dose of ibrutinib is about 980 mg/day. In someembodiments, a dose of ibrutinib is about 1120 mg/day. In someembodiments, a dose of ibrutinib is about 1260 mg/day. In someembodiments, a dose of ibrutinib is about 1400 mg/day. In someembodiments, the pharmaceutical composition further comprisesco-administering an additional therapeutic agent. In some embodiments,the additional therapeutic agent is an anti-HER2 therapeutic agent. Insome embodiments, the anti-HER2 therapeutic agent is selected from thegroup consisting of: trastuzumab, trastuzumab emtansine, pertuzumab,lapatinib, and MM-111 (Merrimack Pharmaceuticals). In some embodiments,the anti-HER2 therapeutic agent is trastuzumab. In some embodiments, theanti-HER2 therapeutic agent is trastuzumab emtansine. In someembodiments, the anti-HER2 therapeutic agent is lapatinib. In someembodiments, the anti-HER2 therapeutic agent is pertuzumab. In someembodiments, the anti-HER2 therapeutic agent is MM-111. In someembodiments, the additional therapeutic agent is a pan-ErbB inhibitor.In some embodiments, the pan-ErbB inhibitor is selected from the groupconsisting of: afatinib, neratinib, and dacomitinib. In someembodiments, the pan-ErbB inhibitor is afatinib. In some embodiments,the pan-ErbB inhibitor is neratinib. In some embodiments, the pan-ErbBinhibitor is dacomitinib. In some embodiments, the additionaltherapeutic agent is an anti-VEGF therapeutic agent. In someembodiments, the anti-VEGF therapeutic agent is selected from the groupconsisting of: bevacizumab, ranibizumab, lapatinib, sunitinib,sorafenib, axitinib, and pazopanib. In some embodiments, the anti-VEGFtherapeutic agent is bevacizumab. In some embodiments, the anti-VEGFtherapeutic agent is ranibizumab. In some embodiments, the anti-VEGFtherapeutic agent is lapatinib. In some embodiments, the anti-VEGFtherapeutic agent is sunitinib. In some embodiments, the anti-VEGFtherapeutic agent is sorafenib. In some embodiments, the anti-VEGFtherapeutic agent is axitinib. In some embodiments, the anti-VEGFtherapeutic agent is pazopanib. In some embodiments, the additionaltherapeutic agent is selected from the group consisting of:temsirolimus, paclitaxel, ASLAN001 (also, ARRY-543, ASLANPharmaceuticals), vorinostat, doxorubicin, cyclophosphamide, cisplatin,docetaxel, and dasatinib. In some embodiments, the additionaltherapeutic agent is doxorubicin. In some embodiments, the additionaltherapeutic agent is docetaxel. In some embodiments, the additionaltherapeutic agent is paclitaxal. In some embodiments, the additionaltherapeutic agent is trastuzumab and docetaxel. In some embodiments, theadditional therapeutic agent is pertuzumab and docetaxel. In someembodiments, the additional therapeutic agent is doxorubicin,cyclophosphamide and paclitaxal. In some embodiments, the additionaltherapeutic agent is doxorubicin, cyclophosphamide and 5-FU. In someembodiments, the HER2-amplified breast cancer is metastatic. In someembodiments, the HER2-amplified breast cancer has metastasized to thebrain. In some embodiments, the HER2-amplified breast cancer isrefractory to treatment. In some embodiments, the HER2-amplified breastcancer is refractory to a treatment selected from: trastuzumab,trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. In someembodiments, the HER2-amplified breast cancer is refractory totrastuzumab. In some embodiments, the HER2-amplified breast cancer isrecurrent. In some embodiments, the HER2-amplified breast cancer has aHER2:CEP17 ratio >4.0. In some embodiments, the HER2-amplified breastcancer has a HER2:CEP17 ratio of 2.2-4.0. In some embodiments, theHER2-amplified breast cancer is graded 3+ using IHC.

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions for use in treating HER2 amplified cancer in a patient,wherein the pharmaceutical composition comprises(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

In some embodiments, the HER2-amplified cancer is selected from thegroup consisting of: breast, colon, endometrial, cervical, urothelial,lung (including, non-small cell lung cancer), ovarian, gastric,gastroesophageal junction (GEJ), head and neck, biliary tract, prostate,and pancreatic cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified breast cancer. In some embodiments, the HER2-amplifiedcancer is HER2-amplified colon cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified endometrial cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified cervicalcancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedurothelial cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified lung cancer. In some embodiments, the HER2-amplified lungcancer is HER2-amplified non-small cell lung cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified ovarian cancer.In some embodiments, the HER2-amplified cancer is HER2-amplified gastriccancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedgastroesophageal junction (GEJ) cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified head and neck cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified biliary tractcancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedprostate cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified pancreatic cancer. In some embodiments, theHER2-amplified cancer is metastatic. In some embodiments, theHER2-amplified cancer has metastasized to the brain. In someembodiments, the HER2-amplified cancer has a HER2:CEP17 ratio >4.0. Insome embodiments, the HER2-amplified cancer has a HER2:CEP17 ratio of2.2-4.0. In some embodiments, the HER2-amplified cancer is graded 3+using IHC. In some embodiments, the HER2-amplified cancer is refractoryto treatment. In some embodiments, the treatment to which theHER2-amplified cancer is refractory is selected from: trastuzumab,trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. In someembodiments, the treatment to which the HER2-amplified cancer isrefractory is trastuzumab. In some embodiments, the HER2-amplifiedcancer is recurrent. In some embodiments, the pharmaceutical compositionfurther comprises co-administering an additional therapeutic agent. Insome embodiments, the additional therapeutic agent is an anti-HER2therapeutic agent. In some embodiments, the anti-HER2 therapeutic agentis selected from the group consisting of: trastuzumab, trastuzumabemtansine, pertuzumab, lapatinib, and MM-111 (MerrimackPharmaceuticals). In some embodiments, the anti-HER2 therapeutic agentis trastuzumab. In some embodiments, the anti-HER2 therapeutic agent istrastuzumab emtansine. In some embodiments, the anti-HER2 therapeuticagent is lapatinib. In some embodiments, the anti-HER2 therapeutic agentis pertuzumab. In some embodiments, the anti-HER2 therapeutic agent isMM-111. In some embodiments, the additional therapeutic agent is apan-ErbB inhibitor. In some embodiments, the pan-ErbB inhibitor isselected from the group consisting of: afatinib, neratinib, anddacomitinib. In some embodiments, the pan-ErbB inhibitor is afatinib. Insome embodiments, the pan-ErbB inhibitor is neratinib. In someembodiments, the pan-ErbB inhibitor is dacomitinib. In some embodiments,the additional therapeutic agent is an anti-VEGF therapeutic agent. Insome embodiments, the anti-VEGF therapeutic agent is selected from thegroup consisting of: bevacizumab, ranibizumab, lapatinib, sunitinib,sorafenib, axitinib, and pazopanib. In some embodiments, the anti-VEGFtherapeutic agent is bevacizumab. In some embodiments, the anti-VEGFtherapeutic agent is ranibizumab. In some embodiments, the anti-VEGFtherapeutic agent is lapatinib. In some embodiments, the anti-VEGFtherapeutic agent is sunitinib. In some embodiments, the anti-VEGFtherapeutic agent is sorafenib. In some embodiments, the anti-VEGFtherapeutic agent is axitinib. In some embodiments, the anti-VEGFtherapeutic agent is pazopanib. In some embodiments, the additionaltherapeutic agent is selected from the group consisting of:temsirolimus, paclitaxel, ASLAN001 (also, ARRY-543, ASLANPharmaceuticals), vorinostat, doxorubicin, cyclophosphamide, cisplatin,docetaxel, and dasatinib. In some embodiments, the additionaltherapeutic agent is doxorubicin. In some embodiments, the additionaltherapeutic agent is docetaxel. In some embodiments, the additionaltherapeutic agent is paclitaxal. In some embodiments, the additionaltherapeutic agent is trastuzumab and docetaxel. In some embodiments, theadditional therapeutic agent is pertuzumab and docetaxel. In someembodiments, the additional therapeutic agent is doxorubicin,cyclophosphamide and paclitaxal. In some embodiments, the additionaltherapeutic agent is doxorubicin, cyclophosphamide and 5-FU. In someembodiments, a dose of ibrutinib is from about 100 mg/day up to, andincluding, about 2000 mg/day. In some embodiments, a dose of ibrutinibis from about 140 mg/day up to, and including, about 840 mg/day. In someembodiments, a dose of ibrutinib is from about 420 mg/day up to, andincluding, about 840 mg/day. In some embodiments, a dose of ibrutinib isabout 140 mg/day. In some embodiments, a dose of ibrutinib is about 280mg/day. In some embodiments, a dose of ibrutinib is about 420 mg/day. Insome embodiments, a dose of ibrutinib is about 560 mg/day. In someembodiments, a dose of ibrutinib is about 700 mg/day. In someembodiments, a dose of ibrutinib is about 840 mg/day. In someembodiments, a dose of ibrutinib is about 980 mg/day. In someembodiments, a dose of ibrutinib is about 1120 mg/day. In someembodiments, a dose of ibrutinib is about 1260 mg/day. In someembodiments, a dose of ibrutinib is about 1400 mg/day.

Disclosed herein, in certain embodiments, are methods for treating HER2amplified breast cancer in an individual in need thereof comprisingadministering to an individual in need thereof a composition comprising(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

In some embodiments, a dose of ibrutinib is from about 100 mg/day up to,and including, about 2000 mg/day. In some embodiments, a dose ofibrutinib is from about 140 mg/day up to, and including, about 840mg/day. In some embodiments, a dose of ibrutinib is from about 420mg/day up to, and including, about 840 mg/day. In some embodiments, adose of ibrutinib is about 140 mg/day. In some embodiments, a dose ofibrutinib is about 280 mg/day. In some embodiments, a dose of ibrutinibis about 420 mg/day. In some embodiments, a dose of ibrutinib is about560 mg/day. In some embodiments, a dose of ibrutinib is about 700mg/day. In some embodiments, a dose of ibrutinib is about 840 mg/day. Insome embodiments, a dose of ibrutinib is about 980 mg/day. In someembodiments, a dose of ibrutinib is about 1120 mg/day. In someembodiments, a dose of ibrutinib is about 1260 mg/day. In someembodiments, a dose of ibrutinib is about 1400 mg/day. In someembodiments, the HER2-amplified breast cancer is metastatic. In someembodiments, the HER2-amplified breast cancer has metastasized to thebrain. In some embodiments, the HER2-amplified breast cancer isrefractory to treatment. In some embodiments, the HER2-amplified breastcancer is refractory to a treatment selected from: trastuzumab,trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. In someembodiments, the HER2-amplified breast cancer is refractory totrastuzumab. In some embodiments, the HER2-amplified breast cancer isrecurrent. In some embodiments, the HER2-amplified breast cancer has aHER2:CEP17 ratio >4.0. In some embodiments, the HER2-amplified breastcancer has a HER2:CEP17 ratio of 2.2-4.0. In some embodiments, theHER2-amplified breast cancer is graded 3+ using IHC. In someembodiments, the methods further comprise co-administering an additionaltherapeutic agent. In some embodiments, the additional therapeutic agentis an anti-HER2 therapeutic agent. In some embodiments, the anti-HER2therapeutic agent is a kinase inhibitor. In some embodiments, theanti-HER2 therapeutic agent is selected from the group consisting of:trastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, and MM-111(Merrimack Pharmaceuticals). In some embodiments, the anti-HER2therapeutic agent is trastuzumab. In some embodiments, the anti-HER2therapeutic agent is trastuzumab emtansine. In some embodiments, theanti-HER2 therapeutic agent is lapatinib. In some embodiments, theanti-HER2 therapeutic agent is pertuzumab. In some embodiments, theanti-HER2 therapeutic agent is MM-111. In some embodiments, theadditional therapeutic agent is a pan-ErbB inhibitor. In someembodiments, the pan-ErbB inhibitor is selected from the groupconsisting of: afatinib, neratinib, and dacomitinib. In someembodiments, the pan-ErbB inhibitor is afatinib. In some embodiments,the pan-ErbB inhibitor is neratinib. In some embodiments, the pan-ErbBinhibitor is dacomitinib. In some embodiments, the additionaltherapeutic agent is an anti-VEGF therapeutic agent. In someembodiments, the anti-VEGF therapeutic agent is selected from the groupconsisting of: bevacizumab, ranibizumab, lapatinib, sunitinib,sorafenib, axitinib, and pazopanib. In some embodiments, the anti-VEGFtherapeutic agent is bevacizumab. In some embodiments, the anti-VEGFtherapeutic agent is ranibizumab. In some embodiments, the anti-VEGFtherapeutic agent is lapatinib. In some embodiments, the anti-VEGFtherapeutic agent is sunitinib. In some embodiments, the anti-VEGFtherapeutic agent is sorafenib. In some embodiments, the anti-VEGFtherapeutic agent is axitinib. In some embodiments, the anti-VEGFtherapeutic agent is pazopanib. In some embodiments, the additionaltherapeutic agent is selected from the group consisting of:temsirolimus, paclitaxel, ASLAN001 (also, ARRY-543, ASLANPharmaceuticals), vorinostat, doxorubicin, cyclophosphamide, cisplatin,docetaxel, and dasatinib. In some embodiments, the additionaltherapeutic agent is doxorubicin. In some embodiments, the additionaltherapeutic agent is docetaxel. In some embodiments, the additionaltherapeutic agent is paclitaxal. In some embodiments, the additionaltherapeutic agent is trastuzumab and docetaxel. In some embodiments, theadditional therapeutic agent is pertuzumab and docetaxel. In someembodiments, the additional therapeutic agent is doxorubicin,cyclophosphamide and paclitaxal. In some embodiments, the additionaltherapeutic agent is doxorubicin, cyclophosphamide and 5-FU.

Disclosed herein, in certain embodiments, are methods for treating HER2amplified cancer in an individual in need thereof comprisingadministering to an individual in need thereof a composition comprising(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

In some embodiments, a dose of ibrutinib is from about 100 mg/day up to,and including, about 2000 mg/day. In some embodiments, a dose ofibrutinib is from about 140 mg/day up to, and including, about 840mg/day. In some embodiments, a dose of ibrutinib is from about 420mg/day up to, and including, about 840 mg/day. In some embodiments, adose of ibrutinib is about 140 mg/day. In some embodiments, a dose ofibrutinib is about 280 mg/day. In some embodiments, a dose of ibrutinibis about 420 mg/day. In some embodiments, a dose of ibrutinib is about560 mg/day. In some embodiments, a dose of ibrutinib is about 700mg/day. In some embodiments, a dose of ibrutinib is about 840 mg/day. Insome embodiments, a dose of ibrutinib is about 980 mg/day. In someembodiments, a dose of ibrutinib is about 1120 mg/day. In someembodiments, a dose of ibrutinib is about 1260 mg/day. In someembodiments, a dose of ibrutinib is about 1400 mg/day. In someembodiments, the HER2-amplified cancer is selected from the groupconsisting of: breast, colon, endometrial, cervical, urothelial, lung(including, non-small cell lung cancer), ovarian, gastric,gastroesophageal junction (GEJ), head and neck, biliary tract, prostate,and pancreatic cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified breast cancer. In some embodiments, the HER2-amplifiedcancer is HER2-amplified colon cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified endometrial cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified cervicalcancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedurothelial cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified lung cancer. In some embodiments, the HER2-amplified lungcancer is HER2-amplified non-small cell lung cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified ovarian cancer.In some embodiments, the HER2-amplified cancer is HER2-amplified gastriccancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedgastroesophageal junction (GEJ) cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified head and neck cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified biliary tractcancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedprostate cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified pancreatic cancer. In some embodiments, theHER2-amplified cancer is metastatic. In some embodiments, theHER2-amplified cancer has metastasized to the brain. In someembodiments, the HER2-amplified cancer has a HER2:CEP17 ratio >4.0. Insome embodiments, the HER2-amplified cancer has a HER2:CEP17 ratio of2.2-4.0. In some embodiments, the HER2-amplified cancer is graded 3+using IHC. In some embodiments, the HER2-amplified cancer is refractoryto treatment. In some embodiments, the treatment to which theHER2-amplified cancer is refractory is selected from: trastuzumab,trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. In someembodiments, the treatment to which the HER2-amplified cancer isrefractory is trastuzumab. In some embodiments, the HER2-amplifiedcancer is recurrent. In some embodiments, the methods further compriseco-administering an additional therapeutic agent. In some embodiments,the additional therapeutic agent is an anti-HER2 therapeutic agent. Insome embodiments, the anti-HER2 therapeutic agent is a kinase inhibitor.In some embodiments, the anti-HER2 therapeutic agent is selected fromthe group consisting of: trastuzumab, trastuzumab emtansine, pertuzumab,lapatinib, and MM-111 (Merrimack Pharmaceuticals). In some embodiments,the anti-HER2 therapeutic agent is trastuzumab. In some embodiments, theanti-HER2 therapeutic agent is trastuzumab emtansine. In someembodiments, the anti-HER2 therapeutic agent is lapatinib. In someembodiments, the anti-HER2 therapeutic agent is pertuzumab. In someembodiments, the anti-HER2 therapeutic agent is MM-111. In someembodiments, the additional therapeutic agent is a pan-ErbB inhibitor.In some embodiments, the pan-ErbB inhibitor is selected from the groupconsisting of: afatinib, neratinib, and dacomitinib. In someembodiments, the pan-ErbB inhibitor is afatinib. In some embodiments,the pan-ErbB inhibitor is neratinib. In some embodiments, the pan-ErbBinhibitor is dacomitinib. In some embodiments, the additionaltherapeutic agent is an anti-VEGF therapeutic agent. In someembodiments, the anti-VEGF therapeutic agent is selected from the groupconsisting of: bevacizumab, ranibizumab, lapatinib, sunitinib,sorafenib, axitinib, and pazopanib. In some embodiments, the anti-VEGFtherapeutic agent is bevacizumab. In some embodiments, the anti-VEGFtherapeutic agent is ranibizumab. In some embodiments, the anti-VEGFtherapeutic agent is lapatinib. In some embodiments, the anti-VEGFtherapeutic agent is sunitinib. In some embodiments, the anti-VEGFtherapeutic agent is sorafenib. In some embodiments, the anti-VEGFtherapeutic agent is axitinib. In some embodiments, the anti-VEGFtherapeutic agent is pazopanib. In some embodiments, the additionaltherapeutic agent is selected from the group consisting of:temsirolimus, paclitaxel, ASLAN001 (also, ARRY-543, ASLANPharmaceuticals), vorinostat, doxorubicin, cyclophosphamide, cisplatin,docetaxel, and dasatinib. In some embodiments, the additionaltherapeutic agent is doxorubicin. In some embodiments, the additionaltherapeutic agent is docetaxel. In some embodiments, the additionaltherapeutic agent is paclitaxal. In some embodiments, the additionaltherapeutic agent is trastuzumab and docetaxel. In some embodiments, theadditional therapeutic agent is pertuzumab and docetaxel. In someembodiments, the additional therapeutic agent is doxorubicin,cyclophosphamide and paclitaxal. In some embodiments, the additionaltherapeutic agent is doxorubicin, cyclophosphamide and 5-FU.

Other objects, features and advantages of the methods and compositionsdescribed herein will become apparent from the following detaileddescription. It should be understood, however, that the detaileddescription and the specific examples, while indicating specificembodiments, are given by way of illustration only. The section headingsused herein are for organizational purposes only and are not to beconstrued as limiting the subject matter described.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: exemplifies the homology among members of the ErbB kinase andTEC kinase families and IC50 values for inhibition of each kinase byibrutinib.

FIG. 2A-FIG. 2D: exemplify the effects of ibrutinib on relative cellgrowth (A) BT-474 cells—ibrutinib, AVL-292 and PCI-45468; (B) SK-BR3cells—ibrutinib, AVL-292 and PCI-4546; (C) UACC-893 cells—ibrutinib; and(D) MDA-MB-453 cells—ibrutinib, AVL-292 and PCI-4546.

FIG. 3A-FIG. 3B: exemplify the effects of ibrutinib on relative cellgrowth after 1 hour ibrutinib incubation (Day 1) followed by wash outand six days culture (Day 6) (A) BT-474 cells—ibrutinib (0.4 μM, 0.25μM, 0.5 μM); (B) SK-BR3 cells—ibrutinib (0.4 μM, 0.25 μM, 0.5 μM).

FIG. 4A-FIG. 4B: exemplify cells with 1 h exposure to ibrutinib had G1arrest as measured at 24 h for both (A) BT-474 cells and (B) SK-BR3cells.

FIG. 5A-FIG. 5C: exemplify the effects of ibrutinib and PCI-24781 onBT-474 cell apoptosis. (A) One hour of ibrutinib treatment (Day 1)followed by followed by wash out and six days culture (Day 6). Apoptosiswas measured as percentage of cells in sub G0. (B, C) three dayscontinuous ibrutinib and PCI-24781 incubation with or without thecaspase inhibitor Q-VD-OPH. Apoptosis was measured as percentage ofcells (B) with annexin-V positivity or (C) in sub G0. *p<0.05; **:p<0.01.

FIG. 6A-FIG. 6C: exemplify the effects of ibrutinib on tumorspheregrowth after 1 hour ibrutinib treatment followed by wash out and sevendays culture. (A) Graph of tumorsphere size for increasingconcentrations of ibrutinib (0.4 μM, 0.25 μM, 0.5 μM) *: p, 0.05; **:p<0.01. (B) Graph of tumorsphere size for each concentration ofibrutinib tested. (C) Photomicrograph of selected tumorspheres for eachconcentration of ibrutinib tested.

FIG. 7: exemplifies the effects of ibrutinib on tumor volume of aMDA-MB-453 mouse xenograft.

FIG. 8: exemplifies gene expression levels of various biomarkers inmultiple cancer cell lines with (+) or without (−) addition of 0.1 μMibrutinib (1 hour treatment).

FIG. 9: exemplifies gene expression levels of various biomarkers inmultiple cancer cell lines with (+) or without (−) addition of 0.1 μMibrutinib (1 hour treatment).

FIG. 10: exemplifies gene expression levels of various biomarkers inBT474 (left panel) and SKBR-3 (right panel) breast cancer cell lineswith (+) or without (−) addition of 0.1 μM or 0.5 μM of ibrutinib, at15, 30 or 2 hour treatment, and with (+) or without (−) washout.

FIG. 11: exemplifies the effects of ibrutinib on expression of theprogenitor cell marker aldehyde dehydrogenase (ALDH) by Aldefluor assay.BT-474 breast cancer cells were treated with 0, 0.1 μM, 0.25 μM 0.5 μMibrutinib for 1 h followed by washout and three days continued cultureor 0.1 μM ibrutinib continuously for three days.

FIG. 12: is an exemplary dose response comparison of the effects ofincreasing concentrations of gefitinib (Iressa) and ibrutinib onMDA-MB-453 cells.

FIG. 13A-FIG. 13B: exemplify the dose response effects of ibrutinib,dacomitinib, neratinib, PCI-45466, lapatinib, and gefitinib on relativecell growth of (A) SK-BR3 cells and (B) MDA-MB-453 cells.

FIG. 14A-FIG. 14C: exemplify the dose response effects of ibrutinib,lapatinib, neratinib, dacomitinib, afatinib, and gefitinib on relativecell growth of (A) BT-474 cells, (B) SK-BR3 cells and (C) MDA-MB-453cells.

FIG. 15A-FIG. 15C: exemplify the dose response effects of ibrutinib,AVL-292 and PCI-45468 on relative cell growth of (A) SK-BR3 cells, (B)MDA-MB-453 and (C) BT-474 cells.

FIG. 16: exemplifies the effects of ibrutinib, AVL-292 and PCI-45468 onHER2 and HER activation and downstream pathways in BT-474 cells.

FIG. 17: exemplifies the effect of ibrutinib on BT-474 cell apoptosis.Ibrutinib was incubated for three days continuously with or without thecaspase inhibitor Q-VD-OPH.

FIG. 18A and FIG. 18B: exemplify the effects of ibrutinib on expressionof the progenitor cell marker aldehyde dehydrogenase (ALDH) by Aldefluorassay (A) and propidium iodide (PI) assay (B) *: p=0.06.

FIG. 19A and FIG. 19B: exemplify the comparison of signaling pathwayinhibition between ibrutinib and afatinib (A) and the associated IC50values (B).

FIG. 20: exemplifies the signaling pathway inhibition of neratinib anddacomitinib. Dacomitinib is less effective at inhibiting the signalingpathways.

FIG. 21: exemplifies ibrutinib as a more potent inhibitor of MEK and Aktpathways than the EGFR and HER2 pathways.

FIG. 22A-FIG. 22C: exemplify the concentrations of ibrutinib to overcomeresistance induced by heregulin in BT-474 cells (A) and MDA-MB-453 cells(B). C illustrates ibrutinib inhibition as assessed by phosphorylationof HER2 and additional downstream targets in the presence or absence ofheregulin.

FIG. 23: exemplifies ibrutinib inhibition on several signaling pathwaysin B cells. Ibrutinib selectively inhibits the Btk signaling pathway inMino cells.

FIG. 24: exemplifies ibrutinib as covalently binds to Btk.

FIG. 25A-FIG. 25C: exemplify BTK dialysis experiment. Ibrutinibirreversibly inhibits Btk.

FIG. 26A-FIG. 26C: exemplify LCK dialysis experiment. Ibrutinibreversibly inhibits LCK.

FIG. 27A-FIG. 27C: exemplify EFGR dialysis experiment. Ibrutinibirreversibly inhibits EFGR.

FIG. 28A-FIG. 28C: exemplify HER4 dialysis experiment. Ibrutinibirreversibly inhibits HER4.

FIG. 29A-FIG. 29D: exemplify time-dependent inhibition of BTK byibrutinib after rapid dilution. FIG. 29A illustrates the progress curvesprofile after pre-incubation. FIG. 29B illustrates the initial velocitytable. FIG. 29C illustrates a graphical representation of preincubationtime vs. initial velocity. FIG. 29D illustrates the parametersassociated with the time-dependent inhibition.

FIG. 30A-FIG. 30D: exemplify time-dependent inhibition of HER2 byibrutinib after rapid dilution. FIG. 30A illustrates the progress curvesprofile after pre-incubation. FIG. 30B illustrates the initial velocitytable. FIG. 30C illustrates a graphical representation of preincubationtime vs. initial velocity. FIG. 30D illustrates the parametersassociated with the time-dependent inhibition.

FIG. 31A-FIG. 31D: exemplify the kinetics of BTK inhibition byibrutinib. FIG. 31A illustrates the progress curves profile at thedifferent concentrations of ibrutinib. FIG. 31B illustrates theparameters associated with the time-dependent inhibition. FIG. 31Cillustrates a graphical representation of concentration vs Kobs. FIG.31D illustrates the Kobs values for each concentration of ibrutinib. FITwas calculated using the equation:FIT=Vs*t+((Vo−Vs)/Kobs)*(1−exp(−Kobs*t)). Kobs was calculated using theequation: Kobs=Kinact*[I]/([I]+Ki*(1+[S]/Km)).

FIG. 32A-FIG. 32D: exemplify the kinetics of HER2 inhibition byibrutinib. FIG. 32A illustrates the progress curves profile at thedifferent concentrations of ibrutinib. FIG. 32B illustrates theparameters associated with the time-dependent inhibition. FIG. 32Cillustrates a graphical representation of concentration vs Kobs. FIG.32D illustrates the Kobs values for each concentration of ibrutinib. FITwas calculated using the equation:FIT=Vs*t+((Vo−Vs)/Kobs)*(1−exp(−Kobs*t)). Kobs was calculated using theequation: Kobs=Kinact*[I]/([I]+Ki*(1+[S]/Km)).

FIG. 33: exemplifies one hour treatment of ibrutinib followed by washouton signaling pathways. Ibrutinib has a long-lasting inhibitory effecteven after day 6 on HER signaling pathway.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein, in certain embodiments, are methods and pharmaceuticalcompositions for treating HER2 amplified cancer in an individual in needthereof comprising administering to an individual in need thereof acomposition comprising(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

Further disclosed herein, in certain embodiments, are methods andpharmaceutical compositions for treating HER2 amplified cancer in anindividual in need thereof comprising administering to an individual inneed thereof a composition comprising(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one

CERTAIN TERMINOLOGY

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include”, “includes,” and “included,” is not limiting.

As used herein, “ACK” and “Accessible Cysteine Kinase” are synonyms.They mean a kinase with an accessible cysteine residue. ACKs include,but are not limited to, BTK, ITK, BMX/ETK, TEC, EFGR, HER2, HER4, LCK,BLK, C-src, FGR, Fyn, HCK, Lyn, YES, ABL, Brk, CSK, FER, JAK3, SYK. Insome embodiments, the ACK is HER2. In some embodiments, the ACK is HER4.

As used herein, “amelioration” refers to any lessening of severity,delay in onset, slowing of growth, slowing of metastasis, or shorteningof duration of HER2-amplified breast cancer, whether permanent ortemporary, lasting or transient that can be attributed to or associatedwith administration of the compound or composition.

The term “Bruton's tyrosine kinase,” as used herein, refers to Bruton'styrosine kinase from Homo sapiens, as disclosed in, e.g., U.S. Pat. No.6,326,469 (GenBank Accession No. NP_000052).

The term “Bruton's tyrosine kinase homolog,” as used herein, refers toorthologs of Bruton's tyrosine kinase, e.g., the orthologs from mouse(GenBank Accession No. AAB47246), dog (GenBank Accession No. XP_549139),rat (GenBank Accession No. NP_001007799), chicken (GenBank Accession No.NP_989564), or zebra fish (GenBank Accession No. XP_698117), and fusionproteins of any of the foregoing that exhibit kinase activity towardsone or more substrates of Bruton's tyrosine kinase (e.g. a peptidesubstrate having the amino acid sequence “AVLESEEELYSSARQ”).

The term “HER2”, also known as ERBB2, also known as “V-erb-b2erythroblastic leukemia viral oncogene homolog 2” means either (a) thenucleic acid sequence encoding a receptor tyrosine kinase that is amember of the epidermal growth factor receptor subfamily, or (b) theprotein thereof. For the nucleic acid sequence that comprises the humanHER2 gene see GenBank Accession No. NM_004448. For the amino acidsequence that comprises the human HER2 protein see GenBank Accession No.NP_004439.

The term “HER4”, also known as ERBB4, also known as “V-erb-b2erythroblastic leukemia viral oncogene homolog 4” means either (a) thenucleic acid sequence encoding a receptor tyrosine kinase that is amember of the epidermal growth factor receptor subfamily, or (b) theprotein thereof. For the nucleic acid sequence that comprises the humanHER4 gene see GenBank Accession No. NM_001042599. For the amino acidsequence that comprises the human HER4 protein see GenBank Accession No.NP_001036064.

The term “homologous cysteine,” as used herein refers to a cysteineresidue found within a sequence position that is homologous to that ofcysteine 481 of Bruton's tyrosine kinase, as defined herein. Forexample, cysteine 482 is the homologous cysteine of the rat ortholog ofBruton's tyrosine kinase; cysteine 479 is the homologous cysteine of thechicken ortholog; and cysteine 481 is the homologous cysteine in thezebra fish ortholog. In another example, the homologous cysteine of TXK,a Tec kinase family member related to Bruton's tyrosine, is Cys350.

The term “irreversible Btk inhibitor,” as used herein, refers to aninhibitor of Btk that can form a covalent bond with an amino acidresidue of Btk. In one embodiment, the irreversible inhibitor of Btk canform a covalent bond with a Cys residue of Btk; in particularembodiments, the irreversible inhibitor can form a covalent bond with aCys 481 residue (or a homolog thereof) of Btk or a cysteine residue inthe homologous corresponding position of another tyrosine kinase, asshown in FIG. 1.

As used herein, the term “pAKT” refers to phosphorylated AKT at Thr308as detected by commercially available phospho-specific antibodies (e.g.Santa Cruz Biotech sc-16646).

As used herein, the term “pERK” refers to phosphorylated ERK1 and ERK2at Thr202/Tyr 204 as detected by commercially available phospho-specificantibodies (e.g. Cell Signaling Technologies #4377).

The terms “individual”, “patient” and “subject” are usedinterchangeable. They refer to a mammal (e.g., a human) which is theobject of treatment, or observation. The term is not to be construed asrequiring the supervision of a medical practitioner (e.g., a physician,physician's assistant, nurse, orderly, hospice care worker).

The terms “treat,” “treating” or “treatment”, as used herein, includelessening of severity of HER2-amplified breast cancer, delay in onset ofHER2-amplified breast cancer, slowing the growth of HER2-amplifiedbreast cancer, slowing metastasis of cells of HER2-amplified breastcancer, shortening of duration of HER2-amplified breast cancer,arresting the development of HER2-amplified breast cancer, causingregression of HER2-amplified breast cancer, relieving a condition causedby of HER2-amplified breast cancer, or stopping symptoms which resultfrom HER2-amplified breast cancer. The terms “treat,” “treating” or“treatment”, include, but are not limited to, prophylactic and/ortherapeutic treatments.

The term “breast cancer”, as used herein, includes ductal carcinoma insitu (intraductal carcinoma), lobular carcinoma in situ, invasive (orinfiltrating) ductal carcinoma, invasive (or infiltrating) lobularcarcinoma, inflammatory breast cancer, triple-negative breast cancer,paget disease of the nipple, phyllodes tumor, angiosarcoma or invasivebreast carcinoma. In some embodiments, the invasive breast carcinoma isfurther categorized into subtypes. In some embodiments, the subtypesinclude adenoid cystic (or adenocystic) carcinoma, low-gradeadenosquamous carcinoma, medullary carcinoma, mucinous (or colloid)carcinoma, papillary carcinoma, tubular carcinoma, metaplasticcarcinoma, micropapillary carcinoma or mixed carcinoma.

In some embodiments, the breast cancer is further classified accordingto stages or how far the tumor cells have spread within the breasttissues and to other portions of the body. In some embodiments, thereare five stages of breast cancer, Stage 0-IV. In some embodiments, Stage0 breast cancer refers to non-invasive breast cancers or that there areno evidence of cancer cells or abnormal non-cancerous cells breaking outof the origin site. In some embodiments, Stage I breast cancer refers toinvasive breast cancer in which the cancer cells have invaded intosurrounding tissues. In some embodiments, Stage I is subclassified intoStage IA and IB, in which Stage IA describes tumor measures up to about2 cm with no spread of cancer cells. Stage IB describes absence of tumorin breast but have small lumps of cancer cells between about 0.2 mm toabout 2 mm within the lymph nodes. In some embodiments, Stage II breastcancer is further subdivided into Stage IIA and IIB. In someembodiments, Stage IIA describes tumor between about 2 cm to about 5 cmin breast only, or absence of tumor in breast but with cancer betweenabout 2 mm to about 2 cm in axillary lymph nodes. In some embodiments,Stage IIB describes tumor larger than about 5 cm in breast only, ortumor between about 2 cm to about 5 cm in breast with presence of smalltumors from about 0.2 mm to about 2 mm in axillary lymph nodes. In someembodiments, Stage III breast cancer is further subdivided into StageIIIA, IIIB, and IIIC. In some embodiments, Stage IIIA describes absenceof tumor or tumor greater than about 5 cm in breast with small tumors inabout 4-9 axillary lymph nodes or small tumors about 0.2 mm to about 2mm in size in axillary lymph nodes. In some embodiments, Stage IIIBdescribes tumor spreading into the chest wall or skin of the breastcausing swelling or ulcer and with presence of tumor in up to about 9axillary lymph nodes. In some embodiments, inflammatory breast cancer isalso considered as Stage IIIB In some embodiments, Stage IIIC describesabsence of tumor or tumor spreading into the chest wall or to the skinof the breast, with tumor present in 10 or more axillary lymph nodes. Insome embodiments, Stage IV breast cancer refers to invasive breastcancer that has metastasized into the lymph nodes and other portions ofthe body.

HER2 Amplified Cancer

Described herein are methods of treating HER2 amplified breast cancer inan individual in need thereof comprising administering to the individuala composition comprising a therapeutically-effective amount of an ACKinhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib). As shown herein, ibrutinib is an ACK inhibitor compound thatis effective in inhibiting the activity of ErbB kinases, such as EGFR(ErbB1), HER2 (ErbB2) and HER4 (ErbB4). Further described herein aremethods of treating HER2 amplified cancer in an individual in needthereof comprising administering to the individual a compositioncomprising a therapeutically-effective amount of an ACK inhibitorcompound (e.g., a BTK inhibitor, such as for example ibrutinib).

HER2 (Human Epidermal Growth Factor Receptor 2) also known as Neu,ErbB2, CD340 (cluster of differentiation 340) and p185 is an epidermalgrowth factor receptor found on cells. The HER2 gene is found on humanchromosome 17. HER2 protein is composed of four plasma membrane-boundreceptor tyrosine kinases. Signaling pathways activated by HER2 proteininclude: mitogen-activated protein kinase (MAPK), phosphoinositide3-kinase (PI3K/Akt), phospholipase C γ, protein kinase C (PKC), andsignal transducer and activator of transcription (STAT).

As used herein, “HER2-amplified cancer” means a cancer (e.g., breastcancer) characterized by amplification (or, over-expression) of the HER2gene. Amplification of HER2 gene expression leads to increased membraneexpression of the HER2 protein. Increased expression is associated withincreased dimerization of HER2 proteins with HER3 and HER4. Increaseddimerization leads to increased activation of the HER2 tyrosine kinaseresulting in excessive mitosis and cell replication.

HER2 amplification has been identified in breast, colon, endometrial,cervical, urothelial, lung (including, non-small cell lung cancer),ovarian, gastric, gastroesophageal junction (GEJ), head and neck,biliary tract, prostate, and pancreatic adenocarcinomas. HER2amplification is present in about 18%-25% of breast cancers. HER2amplification is also present in about 30% of GEJ cancers and about 20%of gastric cancers. In some embodiments, the HER2 amplified cancer isselected from: breast, colon, endometrial, cervical, urothelial, lung,ovarian, salivary duct, gastric and gastroesophageal junction (GEJ)cancer. In some embodiments, the HER2 amplified cancer is HER2 amplifiedbreast cancer. In some embodiments, the HER2 amplified cancer is HER2amplified gastric cancer. In some embodiments, the HER2 amplified canceris HER2 amplified gastroesophageal junction (GEJ) cancer.

HER2 amplified tumors are characterized by an aggressive phenotype(e.g., increased cell proliferation, increased cell survival, increasedcell motility, and increased cell adhesion), increased metastasis,increased relapse, shorter disease-free survival and poorer overallsurvival. In some embodiments, the HER2 amplified cancer is metastaticHER2 amplified cancer. Individuals with HER2 amplified breast cancer areat a high risk of developing brain metastases. In some embodiments, theHER2 amplified breast cancer is metastatic HER2 amplified breast cancer.In some embodiments, the HER2 amplified breast cancer has metastasizedto the brain.

The degree of HER2 amplification is variable amongst tumors. Some tumorshave high levels of amplification (HER2:CEP17 ratio >4.0), whereasothers have lower levels amplification (HER2:CEP17 ratio of 2.0-4.0, orHER2:CEP17 ratio of 2.2-4.0). Several tests are used to diagnose andclassify HER2 amplification: immunohistochemistry (IHC), Fluorescence InSitu Hybridization (FISH), Subtraction Probe Technology Chromogenic InSitu Hybridization (SPoT-Light HER2 CISH), and Inform HER2 Dual In SituHybridization (ISH). Using IHC, a cancer is classified as HER2-amplifiedif it is rated 3+. Using FISH, a cancer is generally classified asHER2-amplified if it has a HER2:CEP17 ratio of 2.2-4.0. A HER2:CEP17ratio of 1.8 to 2.2 is considered equivocal.

In some instances, HER2 amplified tumors is associated with the presenceand in some cases elevated expression of heregulin. Heregulin (HRG, alsoknown as neuregulin) is a member of the EGF-like growth anddifferentiation factors. There are four isoforms of HRG (HRG1-HRG4). HRGbinds with high affinity to the receptors of ErbB3 and ErbB4, members ofthe human epidermal growth factor receptor (EGFR) family of receptors.Upon activation, ErbB3 undergoes heterodimerization with other membersof the ErbB family, leading to cell differentiation, migration,proliferation, and survival.

In some cases, the presence and/or elevated level of HRG in breastcancer is correlated with poor histological grades. In some cases, thepresence and/or elevated level of HRG in HER2 amplified breast cancer iscorrelated with poor histological grades. In some cases, the presenceand/or elevated level of HRG in HER2 amplified tumors is correlated withpoor histological grades.

In some cases, breast cancer contains an elevated level of HRG. In somecases, HER2 amplified breast cancer contains an elevated level of HRG.In some cases, HER2 amplified tumors contains an elevated level of HRG.

In some cases, HRG induces resistance in breast cancer. In some cases,HRG induces resistance in HER2 amplified breast cancer. In some cases,HRG induces resistance in HER2 amplified tumors.

In some cases, the presence or absence or the expression level of HRG isused to select a patient for therapy. In some cases, the presence orabsence or the expression level of HRG is used to monitor a patient'streatment progress. In some cases, the presence or absence or theexpression level of HRG is used to optimize a therapeutic regimen.

Current agents used to treat HER2-amplified cancer include trastuzumab,trastuzumab emtansine (also, ado-trastuzumab emtansine), pertuzumab, andlapatinib. Certain individuals with HER2 amplified breast cancer treatedwith trastuzumab develop recurrent disease, even if placed on adjuvanttrastuzumab therapy. Many patients with HER2 amplified cancer do notrespond to therapy or develop refractory disease, in some instanceswithin 1 year of treatment. In some embodiments, the HER2 amplifiedcancer is recurrent. In some embodiments, the HER2 amplified cancer isrefractory, for example to trastuzumab.

Administration

Described herein are methods and pharmaceutical compositions of treatingHER2 amplified breast cancer in an individual in need thereof comprisingadministering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound (e.g., aBTK inhibitor, such as for example ibrutinib). Further described hereinare methods and pharmaceutical compositions of treating HER2 amplifiedcancer in an individual in need thereof comprising administering to theindividual a composition comprising a therapeutically-effective amountof an ACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib). In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

The ACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) is administered before, during or after the development ofthe HER2-amplified cancer. In some embodiments, the ACK inhibitorcompound (e.g., a BTK inhibitor, such as for example ibrutinib) is usedas a prophylactic and is administered continuously to subjects with apropensity to develop HER2-amplified cancer. In some embodiments, theACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) is administered to an individual during or as soon aspossible after the development of HER2-amplified cancer. In someembodiments, the administration of the ACK inhibitor compound (e.g., aBTK inhibitor, such as for example ibrutinib) is initiated within thefirst 48 hours of the onset of the symptoms, within the first 6 hours ofthe onset of the symptoms, or within 3 hours of the onset of thesymptoms. In some embodiments, the initial administration of the ACKinhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) is via any route practical, such as, for example, anintravenous injection, a bolus injection, infusion over 5 minutes toabout 5 hours, a pill, a capsule, transdermal patch, buccal delivery,and the like, or combination thereof. The ACK inhibitor compound (e.g.,a BTK inhibitor, such as for example ibrutinib) should be administeredas soon as is practicable after the onset of a disorder is detected orsuspected, and for a length of time necessary for the treatment of thedisease, such as, for example, from about 1 month to about 3 months. Thelength of treatment can vary for each subject, and the length can bedetermined using the known criteria. In some embodiments, the ACKinhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) is administered for at least 2 weeks, between about 1 monthto about 5 years, or from about 1 month to about 3 years.

Therapeutically effective amounts will depend on the severity and courseof the disorder, previous therapy, the patient's health status, weight,and response to the drugs, and the judgment of the treating physician.Prophylactically effective amounts depend on the patient's state ofhealth, weight, the severity and course of the disease, previoustherapy, response to the drugs, and the judgment of the treatingphysician.

In some embodiments, the ACK inhibitor compound (e.g., a BTK inhibitor,such as for example ibrutinib) is administered to the patient on aregular basis, e.g., three times a day, two times a day, once a day,every other day or every 3 days. In other embodiments, the ACK inhibitorcompound (e.g., a BTK inhibitor, such as for example ibrutinib) isadministered to the patient on an intermittent basis, e.g., twice a dayfollowed by once a day followed by three times a day; or the first twodays of every week; or the first, second and third day of a week. Insome embodiments, intermittent dosing is as effective as regular dosing.In further or alternative embodiments, the ACK inhibitor compound (e.g.,a BTK inhibitor, such as for example ibrutinib) is administered onlywhen the patient exhibits a particular symptom, e.g., the onset of pain,or the onset of a fever, or the onset of an inflammation, or the onsetof a skin disorder. Dosing schedules of each compound may depend on theother or may be independent of the other.

In the case wherein the patient's condition does not improve, upon thedoctor's discretion the administration of the compounds may beadministered chronically, that is, for an extended period of time,including throughout the duration of the patient's life in order toameliorate or otherwise control or limit the symptoms of the patient'sdisorder.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the compounds may be givencontinuously; alternatively, the dose of drug being administered may betemporarily reduced or temporarily suspended for a certain length oftime (i.e., a “drug holiday”). The length of the drug holiday can varybetween 2 days and 1 year, including by way of example only, 2 days, 3days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days,180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or365 days. The dose reduction during a drug holiday may be from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenanceregimen is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, of the ACK inhibitor compound(e.g., a BTK inhibitor, such as for example ibrutinib) can be reduced,as a function of the symptoms, to a level at which the individual'simproved condition is retained. Individuals can, however, requireintermittent treatment on a long-term basis upon any recurrence ofsymptoms.

The amount of the ACK inhibitor compound (e.g., a BTK inhibitor, such asfor example ibrutinib) will vary depending upon factors such as theparticular compound, disorder and its severity, the identity (e.g.,weight) of the subject or host in need of treatment, and is determinedaccording to the particular circumstances surrounding the case,including, e.g., the specific agents being administered, the routes ofadministration, and the subject or host being treated. In general,however, doses employed for adult human treatment will typically be inthe range of 0.02-5000 mg per day, or from about 1-1500 mg per day. Thedesired dose may be presented in a single dose or as divided dosesadministered simultaneously (or over a short period of time) or atappropriate intervals, for example as two, three, four or more sub-dosesper day.

In some embodiments, the therapeutic amount of the ACK inhibitor (e.g.,a BTK inhibitor, such as for example ibrutinib) is from 10 mg/day up to,and including, 2000 mg/day. In some embodiments, the therapeutic amountof the ACK inhibitor (e.g., a BTK inhibitor, such as for exampleibrutinib) is from 40 mg/day up to, and including, 2000 mg/day. In someembodiments, the therapeutic amount of the ACK inhibitor (e.g., a BTKinhibitor, such as for example ibrutinib) is from 100 mg/day up to, andincluding, 2000 mg/day. In some embodiments, the amount of the ACKinhibitor (e.g., a BTK inhibitor, such as for example ibrutinib) is from140 mg/day up to, and including, 840 mg/day. In some embodiments, theamount of the ACK inhibitor (e.g., a BTK inhibitor, such as for exampleibrutinib) is from 420 mg/day up to, and including, 840 mg/day. In someembodiments, the amount of the ACK inhibitor (e.g. a BTK inhibitor, suchas for example ibrutinib) is about 10 mg/day, about 11 mg/day, about 12mg/day, about 13 mg/day, about 14 mg/day, about 15 mg/day, about 16mg/day, about 17 mg/day, about 18 mg/day, about 19 mg/day, about 20mg/day, about 25 mg/day, about 30 mg/day, about 35 mg/day, about 40mg/day, about 45 mg/day, about 50 mg/day, about 55 mg/day, about 60mg/day, about 65 mg/day, about 70 mg/day, about 75 mg/day, about 80mg/day, about 85 mg/day, about 90 mg/day, about 95 mg/day, about 100mg/day, about 110 mg/day, about 120 mg/day, about 125 mg/day, about 130mg/day, about 135 mg/day, about 140 mg/day, about 150 mg/day, about 160mg/day, about 170 mg/day, about 180 mg/day, about 190 mg/day, about 200mg/day, about 280 mg/day, about 360 mg/day, about 420 mg/day, about 560mg/day, about 700 mg/day, about 840 mg/day, about 980 mg/day, about 1120mg/day, or about 1260 mg/day. In some embodiments, the amount of the ACKinhibitor (e.g., a BTK inhibitor, such as for example ibrutinib) isabout 140 mg/day. In some embodiments, the amount of the ACK inhibitor(e.g., a BTK inhibitor, such as for example ibrutinib) is about 280mg/day. In some embodiments, the amount of the ACK inhibitor (e.g., aBTK inhibitor, such as for example ibrutinib) is about 420 mg/day. Insome embodiments, the amount of the ACK inhibitor (e.g., a BTKinhibitor, such as for example ibrutinib) is about 560 mg/day. In someembodiments, the amount of the ACK inhibitor (e.g., a BTK inhibitor,such as for example ibrutinib) is about 700 mg/day. In some embodiments,the amount of the ACK inhibitor (e.g., a BTK inhibitor, such as forexample ibrutinib) is about 840 mg/day. In some embodiments, the amountof the ACK inhibitor (e.g., a BTK inhibitor, such as for exampleibrutinib) is about 980 mg/day. In some embodiments, the amount of theACK inhibitor (e.g., a BTK inhibitor, such as for example ibrutinib) isabout 1120 mg/day. In some embodiments, the amount of the ACK inhibitor(e.g., a BTK inhibitor, such as for example ibrutinib) is about 1260mg/day. In some embodiments, the amount of the ACK inhibitor (e.g., aBTK inhibitor, such as for example ibrutinib) is about 1400 mg/day.

In some embodiments, the dosage of the ACK inhibitor (e.g., a BTKinhibitor, such as for example ibrutinib) is escalated over time. Insome embodiments, the dosage of the ACK inhibitor (e.g., a BTKinhibitor, such as for example ibrutinib) is escalated from at or about1.25 mg/kg/day to at or about 12.5 mg/kg/day over a predetermined periodof time. In some embodiments the predetermined period of time is over 1month, over 2 months, over 3 months, over 4 months, over 5 months, over6 months, over 7 months, over 8 months, over 9 months, over 10 months,over 11 months, over 12 months, over 18 months, over 24 months orlonger.

The ACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) may be formulated into unit dosage forms suitable for singleadministration of precise dosages. In unit dosage form, the formulationis divided into unit doses containing appropriate quantities of one orboth compounds. The unit dosage may be in the form of a packagecontaining discrete quantities of the formulation. Non-limiting examplesare packaged tablets or capsules, and powders in vials or ampoules.Aqueous suspension compositions can be packaged in single-dosenon-reclosable containers. Alternatively, multiple-dose reclosablecontainers can be used, in which case it is typical to include apreservative in the composition. By way of example only, formulationsfor parenteral injection may be presented in unit dosage form, whichinclude, but are not limited to ampoules, or in multi-dose containers,with an added preservative.

It is understood that a medical professional will determine the dosageregimen in accordance with a variety of factors. These factors includethe solid tumor from which the subject suffers, the degree ofmetastasis, as well as the age, weight, sex, diet, and medical conditionof the subject.

Compounds

Described herein are methods and pharmaceutical compositions of treatingHER2 amplified breast cancer in an individual in need thereof comprisingadministering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound (e.g., aBTK inhibitor, such as for example ibrutinib). Further described hereinare methods and pharmaceutical compositions of treating HER2 amplifiedcancer in an individual in need thereof comprising administering to theindividual a composition comprising a therapeutically-effective amountof an ACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib).

Definition of standard chemistry terms are found in reference works,including Carey and Sundberg “ADVANCED ORGANIC CHEMISTRY 4^(TH) ED.”Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwiseindicated, conventional methods of mass spectroscopy, NMR, HPLC, proteinchemistry, biochemistry, recombinant DNA techniques and pharmacology,within the skill of the art are employed. Unless specific definitionsare provided, the nomenclature employed in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those known in the art. Standard techniques are optionallyused for chemical syntheses, chemical analyses, pharmaceuticalpreparation, formulation, and delivery, and treatment of patients.Standard techniques are optionally used for recombinant DNA,oligonucleotide synthesis, and tissue culture and transformation (e.g.,electroporation, lipofection). Reactions and purification techniques areperformed using documented methodologies or as described herein.

It is to be understood that the methods and compositions describedherein are not limited to the particular methodology, protocols, celllines, constructs, and reagents described herein and as such optionallyvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the methods and compositions describedherein, which will be limited only by the appended claims.

Unless stated otherwise, the terms used for complex moieties (i.e.,multiple chains of moieties) are to be read equivalently either fromleft to right or right to left. For example, the groupalkylenecycloalkylene refers both to an alkylene group followed by acycloalkylene group or as a cycloalkylene group followed by an alkylenegroup.

The suffix “ene” appended to a group indicates that such a group is adiradical. By way of example only, a methylene is a diradical of amethyl group, that is, it is a —CH₂— group; and an ethylene is adiradical of an ethyl group, i.e., —CH₂CH₂—.

An “alkyl” group refers to an aliphatic hydrocarbon group. The alkylmoiety includes a “saturated alkyl” group, which means that it does notcontain any alkene or alkyne moieties. The alkyl moiety also includes an“unsaturated alkyl” moiety, which means that it contains at least onealkene or alkyne moiety. An “alkene” moiety refers to a group that hasat least one carbon-carbon double bond, and an “alkyne” moiety refers toa group that has at least one carbon-carbon triple bond. The alkylmoiety, whether saturated or unsaturated, includes branched, straightchain, or cyclic moieties. Depending on the structure, an alkyl groupincludes a monoradical or a diradical (i.e., an alkylene group), and ifa “lower alkyl” having 1 to 6 carbon atoms.

As used herein, C₁-C_(x) includes C₁-C₂, C₁-C₃ . . . C₁-C_(x).

The “alkyl” moiety optionally has 1 to 10 carbon atoms (whenever itappears herein, a numerical range such as “1 to 10” refers to eachinteger in the given range; e.g., “1 to 10 carbon atoms” means that thealkyl group is selected from a moiety having 1 carbon atom, 2 carbonatoms, 3 carbon atoms, etc., up to and including 10 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group of thecompounds described herein may be designated as “C₁-C₄ alkyl” or similardesignations. By way of example only, “C₁-C₄ alkyl” indicates that thereare one to four carbon atoms in the alkyl chain, i.e., the alkyl chainis selected from among methyl, ethyl, propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and t-butyl. Thus C₁-C₄ alkyl includes C₁-C₂ alkyland C₁-C₃ alkyl. Alkyl groups are optionally substituted orunsubstituted. Typical alkyl groups include, but are in no way limitedto, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl,pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and the like.

The term “alkenyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a double bond that is not part of anaromatic group. That is, an alkenyl group begins with the atoms—C(R)═C(R)—R, wherein R refers to the remaining portions of the alkenylgroup, which are either the same or different. The alkenyl moiety isoptionally branched, straight chain, or cyclic (in which case, it isalso known as a “cycloalkenyl” group). Depending on the structure, analkenyl group includes a monoradical or a diradical (i.e., an alkenylenegroup). Alkenyl groups are optionally substituted. Non-limiting examplesof an alkenyl group include —CH═CH₂, —C(CH₃)═CH₂, —CH═CHCH₃,—C(CH₃)═CHCH₃. Alkenylene groups include, but are not limited to,—CH═CH—, —C(CH₃)═CH—, —CH═CHCH₂—, —CH═CHCH₂CH₂— and —C(CH₃)═CHCH₂—.Alkenyl groups optionally have 2 to 10 carbons, and if a “lower alkenyl”having 2 to 6 carbon atoms.

The term “alkynyl” refers to a type of alkyl group in which the firsttwo atoms of the alkyl group form a triple bond. That is, an alkynylgroup begins with the atoms —C≡C—R, wherein R refers to the remainingportions of the alkynyl group, which is either the same or different.The “R” portion of the alkynyl moiety may be branched, straight chain,or cyclic. Depending on the structure, an alkynyl group includes amonoradical or a diradical (i.e., an alkynylene group). Alkynyl groupsare optionally substituted. Non-limiting examples of an alkynyl groupinclude, but are not limited to, —C≡CH, —C≡CCH₃, —C≡CCH₂CH₃, and—C≡CCH₂—. Alkynyl groups optionally have 2 to 10 carbons, and if a“lower alkynyl” having 2 to 6 carbon atoms.

An “alkoxy” group refers to a (alkyl)O— group, where alkyl is as definedherein.

“Hydroxyalkyl” refers to an alkyl radical, as defined herein,substituted with at least one hydroxy group. Non-limiting examples of ahydroxyalkyl include, but are not limited to, hydroxymethyl,2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl,1-(hydroxymethyl)-2-methylpropyl, 2-hydroxybutyl, 3-hydroxybutyl,4-hydroxybutyl, 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl,2,3-dihydroxybutyl, 3,4-dihydroxybutyl and2-(hydroxymethyl)-3-hydroxypropyl.

“Alkoxyalkyl” refers to an alkyl radical, as defined herein, substitutedwith an alkoxy group, as defined herein.

The term “alkylamine” refers to the —N(alkyl)_(x)H_(y) group, where xand y are selected from among x=1, y=1 and x=2, y=0. When x=2, the alkylgroups, taken together with the N atom to which they are attached,optionally form a cyclic ring system.

“Alkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine, as defined herein.

“Hydroxyalkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine, and alkylhydroxy, as defined herein.

“Alkoxyalkylaminoalkyl” refers to an alkyl radical, as defined herein,substituted with an alkylamine and substituted with an alkylalkoxy, asdefined herein.

An “amide” is a chemical moiety with the formula —C(O)NHR or —NHC(O)R,where R is selected from among alkyl, cycloalkyl, aryl, heteroaryl(bonded through a ring carbon) and heteroalicyclic (bonded through aring carbon). In some embodiments, an amide moiety forms a linkagebetween an amino acid or a peptide molecule and a compound describedherein, thereby forming a prodrug. Any amine, or carboxyl side chain onthe compounds described herein can be amidified. The procedures andspecific groups to make such amides are found in sources such as Greeneand Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wiley& Sons, New York, N.Y., 1999, which is incorporated herein by referencefor this disclosure.

The term “ester” refers to a chemical moiety with formula —COOR, where Ris selected from among alkyl, cycloalkyl, aryl, heteroaryl (bondedthrough a ring carbon) and heteroalicyclic (bonded through a ringcarbon). Any hydroxy, or carboxyl side chain on the compounds describedherein can be esterified. The procedures and specific groups to makesuch esters are found in sources such as Greene and Wuts, ProtectiveGroups in Organic Synthesis, 3^(rd) Ed., John Wiley & Sons, New York,N.Y., 1999, which is incorporated herein by reference for thisdisclosure.

As used herein, the term “ring” refers to any covalently closedstructure. Rings include, for example, carbocycles (e.g., aryls andcycloalkyls), heterocycles (e.g., heteroaryls and non-aromaticheterocycles), aromatics (e.g. aryls and heteroaryls), and non-aromatics(e.g., cycloalkyls and non-aromatic heterocycles). Rings can beoptionally substituted. Rings can be monocyclic or polycyclic.

As used herein, the term “ring system” refers to one, or more than onering.

The term “membered ring” can embrace any cyclic structure. The term“membered” is meant to denote the number of skeletal atoms thatconstitute the ring. Thus, for example, cyclohexyl, pyridine, pyran andthiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, andthiophene are 5-membered rings.

The term “fused” refers to structures in which two or more rings shareone or more bonds.

The term “carbocyclic” or “carbocycle” refers to a ring wherein each ofthe atoms forming the ring is a carbon atom. Carbocycle includes aryland cycloalkyl. The term thus distinguishes carbocycle from heterocycle(“heterocyclic”) in which the ring backbone contains at least one atomwhich is different from carbon (i.e. a heteroatom). Heterocycle includesheteroaryl and heterocycloalkyl. Carbocycles and heterocycles can beoptionally substituted.

The term “aromatic” refers to a planar ring having a delocalizedπ-electron system containing 4n+2π electrons, where n is an integer.Aromatic rings can be formed from five, six, seven, eight, nine, or morethan nine atoms. Aromatics can be optionally substituted. The term“aromatic” includes both carbocyclic aryl (e.g., phenyl) andheterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups (e.g.,pyridine). The term includes monocyclic or fused-ring polycyclic (i.e.,rings which share adjacent pairs of carbon atoms) groups.

As used herein, the term “aryl” refers to an aromatic ring wherein eachof the atoms forming the ring is a carbon atom. Aryl rings can be formedby five, six, seven, eight, nine, or more than nine carbon atoms. Arylgroups can be optionally substituted. Examples of aryl groups include,but are not limited to phenyl, naphthalenyl, phenanthrenyl, anthracenyl,fluorenyl, and indenyl. Depending on the structure, an aryl group can bea monoradical or a diradical (i.e., an arylene group).

An “aryloxy” group refers to an (aryl)O— group, where aryl is as definedherein.

The term “carbonyl” as used herein refers to a group containing a moietyselected from the group consisting of —C(O)—, —S(O)—, —S(O)2-, and—C(S)—, including, but not limited to, groups containing a least oneketone group, and/or at least one aldehyde group, and/or at least oneester group, and/or at least one carboxylic acid group, and/or at leastone thioester group. Such carbonyl groups include ketones, aldehydes,carboxylic acids, esters, and thioesters. In some embodiments, suchgroups are a part of linear, branched, or cyclic molecules.

The term “cycloalkyl” refers to a monocyclic or polycyclic radical thatcontains only carbon and hydrogen, and is optionally saturated,partially unsaturated, or fully unsaturated. Cycloalkyl groups includegroups having from 3 to 10 ring atoms. Illustrative examples ofcycloalkyl groups include the following moieties:

and the like. Depending on the structure, a cycloalkyl group is either amonoradical or a diradical (e.g., an cycloalkylene group), and if a“lower cycloalkyl” having 3 to 8 carbon atoms.

“Cycloalkylalkyl” means an alkyl radical, as defined herein, substitutedwith a cycloalkyl group. Non-limiting cycloalkylalkyl groups includecyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl,cyclohexylmethyl, and the like.

The term “heterocycle” refers to heteroaromatic and heteroalicyclicgroups containing one to four heteroatoms each selected from O, S and N,wherein each heterocyclic group has from 4 to 10 atoms in its ringsystem, and with the proviso that the ring of said group does notcontain two adjacent 0 or S atoms. Herein, whenever the number of carbonatoms in a heterocycle is indicated (e.g., C₁-C₆ heterocycle), at leastone other atom (the heteroatom) must be present in the ring.Designations such as “C₁-C₆ heterocycle” refer only to the number ofcarbon atoms in the ring and do not refer to the total number of atomsin the ring. It is understood that the heterocylic ring can haveadditional heteroatoms in the ring. Designations such as “4-6 memberedheterocycle” refer to the total number of atoms that are contained inthe ring (i.e., a four, five, or six membered ring, in which at leastone atom is a carbon atom, at least one atom is a heteroatom and theremaining two to four atoms are either carbon atoms or heteroatoms). Inheterocycles that have two or more heteroatoms, those two or moreheteroatoms can be the same or different from one another. Heterocyclescan be optionally substituted. Binding to a heterocycle can be at aheteroatom or via a carbon atom. Non-aromatic heterocyclic groupsinclude groups having only 4 atoms in their ring system, but aromaticheterocyclic groups must have at least 5 atoms in their ring system. Theheterocyclic groups include benzo-fused ring systems. An example of a4-membered heterocyclic group is azetidinyl (derived from azetidine). Anexample of a 5-membered heterocyclic group is thiazolyl. An example of a6-membered heterocyclic group is pyridyl, and an example of a10-membered heterocyclic group is quinolinyl. Examples of non-aromaticheterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl,homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,3-azabicyclo[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl, 3H-indolyl andquinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl,imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl,furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl,quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl,cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl,triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl,furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl,benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, andfuropyridinyl. The foregoing groups, as derived from the groups listedabove, are optionally C-attached or N-attached where such is possible.For instance, a group derived from pyrrole includes pyrrol-1-yl(N-attached) or pyrrol-3-yl (C-attached). Further, a group derived fromimidazole includes imidazol-1-yl or imidazol-3-yl (both N-attached) orimidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). Theheterocyclic groups include benzo-fused ring systems and ring systemssubstituted with one or two oxo (═O) moieties such as pyrrolidin-2-one.Depending on the structure, a heterocycle group can be a monoradical ora diradical (i.e., a heterocyclene group).

The terms “heteroaryl” or, alternatively, “heteroaromatic” refers to anaromatic group that includes one or more ring heteroatoms selected fromnitrogen, oxygen and sulfur. An N-containing “heteroaromatic” or“heteroaryl” moiety refers to an aromatic group in which at least one ofthe skeletal atoms of the ring is a nitrogen atom. Illustrative examplesof heteroaryl groups include the following moieties:

and the like. Depending on the structure, a heteroaryl group can be amonoradical or a diradical (i.e., a heteroarylene group).

As used herein, the term “non-aromatic heterocycle”, “heterocycloalkyl”or “heteroalicyclic” refers to a non-aromatic ring wherein one or moreatoms forming the ring is a heteroatom. A “non-aromatic heterocycle” or“heterocycloalkyl” group refers to a cycloalkyl group that includes atleast one heteroatom selected from nitrogen, oxygen and sulfur. In someembodiments, the radicals are fused with an aryl or heteroaryl.Heterocycloalkyl rings can be formed by three, four, five, six, seven,eight, nine, or more than nine atoms. Heterocycloalkyl rings can beoptionally substituted. In certain embodiments, non-aromaticheterocycles contain one or more carbonyl or thiocarbonyl groups suchas, for example, oxo- and thio-containing groups. Examples ofheterocycloalkyls include, but are not limited to, lactams, lactones,cyclic imides, cyclic thioimides, cyclic carbamates,tetrahydrothiopyran, 4H-pyran, tetrahydropyran, piperidine, 1,3-dioxin,1,3-dioxane, 1,4-dioxin, 1,4-dioxane, piperazine, 1,3-oxathiane,1,4-oxathiin, 1,4-oxathiane, tetrahydro-1,4-thiazine, 2H-1,2-oxazine,maleimide, succinimide, barbituric acid, thiobarbituric acid,dioxopiperazine, hydantoin, dihydrouracil, morpholine, trioxane,hexahydro-1,3,5-triazine, tetrahydrothiophene, tetrahydrofuran,pyrroline, pyrrolidine, pyrrolidone, pyrrolidione, pyrazoline,pyrazolidine, imidazoline, imidazolidine, 1,3-dioxole, 1,3-dioxolane,1,3-dithiole, 1,3-dithiolane, isoxazoline, isoxazolidine, oxazoline,oxazolidine, oxazolidinone, thiazoline, thiazolidine, and1,3-oxathiolane. Illustrative examples of heterocycloalkyl groups, alsoreferred to as non-aromatic heterocycles, include:

and the like. The term heteroalicyclic also includes all ring forms ofthe carbohydrates, including but not limited to the monosaccharides, thedisaccharides and the oligosaccharides. Depending on the structure, aheterocycloalkyl group can be a monoradical or a diradical (i.e., aheterocycloalkylene group).

The term “halo” or, alternatively, “halogen” or “halide” means fluoro,chloro, bromo and iodo.

The term “haloalkyl,” refers to alkyl structures in which at least onehydrogen is replaced with a halogen atom. In certain embodiments inwhich two or more hydrogen atoms are replaced with halogen atoms, thehalogen atoms are all the same as one another. In other embodiments inwhich two or more hydrogen atoms are replaced with halogen atoms, thehalogen atoms are not all the same as one another.

The term “fluoroalkyl,” as used herein, refers to alkyl group in whichat least one hydrogen is replaced with a fluorine atom. Examples offluoroalkyl groups include, but are not limited to, —CF₃, —CH₂CF₃,—CF₂CF₃, —CH₂CH₂CF₃ and the like.

As used herein, the term “heteroalkyl” refers to optionally substitutedalkyl radicals in which one or more skeletal chain atoms is aheteroatom, e.g., oxygen, nitrogen, sulfur, silicon, phosphorus orcombinations thereof. The heteroatom(s) are placed at any interiorposition of the heteroalkyl group or at the position at which theheteroalkyl group is attached to the remainder of the molecule. Examplesinclude, but are not limited to, —CH₂—O—CH₃, —CH₂—CH₂—O—CH₃,—CH₂—NH—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—N(CH₃)—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)₂—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. In addition, in some embodiments, up to twoheteroatoms are consecutive, such as, by way of example, —CH₂—NH—OCH₃and —CH₂—O—Si(CH₃)₃.

The term “heteroatom” refers to an atom other than carbon or hydrogen.Heteroatoms are typically independently selected from among oxygen,sulfur, nitrogen, silicon and phosphorus, but are not limited to theseatoms. In embodiments in which two or more heteroatoms are present, thetwo or more heteroatoms can all be the same as one another, or some orall of the two or more heteroatoms can each be different from theothers.

The term “bond” or “single bond” refers to a chemical bond between twoatoms, or two moieties when the atoms joined by the bond are consideredto be part of larger substructure.

The term “moiety” refers to a specific segment or functional group of amolecule. Chemical moieties are often recognized chemical entitiesembedded in or appended to a molecule.

A “thioalkoxy” or “alkylthio” group refers to a —S-alkyl group.

A “SH” group is also referred to either as a thiol group or a sulfhydrylgroup.

The term “optionally substituted” or “substituted” means that thereferenced group may be substituted with one or more additional group(s)individually and independently selected from alkyl, cycloalkyl, aryl,heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, alkylthio,arylthio, alkylsulfoxide, arylsulfoxide, alkylsulfone, arylsulfone,cyano, halo, acyl, nitro, haloalkyl, fluoroalkyl, amino, including mono-and di-substituted amino groups, and the protected derivatives thereof.By way of example an optional substituents may be L_(s)R_(s), whereineach L_(s) is independently selected from a bond, —O—, —C(═O)—, —S—,—S(═O)—, —S(═O)₂—, —NH—, —NHC(O)—, —C(O)NH—, S(═O)₂NH—, —NHS(═O)₂,—OC(O)NH—, —NHC(O)O—, -(substituted or unsubstituted C₁-C₆ alkyl), or-(substituted or unsubstituted C₂-C₆ alkenyl); and each R_(s) isindependently selected from H, (substituted or unsubstitutedC₁-C₄alkyl), (substituted or unsubstituted C₃-C₆cycloalkyl), heteroaryl,or heteroalkyl. The protecting groups that form the protectivederivatives of the above substituents include those found in sourcessuch as Greene and Wuts, above.

ACK Inhibitor Compounds

Described herein are methods and pharmaceutical compositions of treatingHER2 amplified breast cancer in an individual in need thereof comprisingadministering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound (e.g., aBTK inhibitor, such as for example ibrutinib). Further described hereinare methods and pharmaceutical compositions of treating HER2 amplifiedcancer in an individual in need thereof comprising administering to theindividual a composition comprising a therapeutically-effective amountof an ACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib).

The ACK inhibitor compounds described herein are selective for kinaseshaving an accessible cysteine that is able to form a covalent bond witha Michael acceptor moiety on the inhibitor compound. In someembodiments, the cysteine residue is accessible or becomes accessiblewhen the binding site moiety of the irreversible inhibitor binds to thekinase. That is, the binding site moiety of the irreversible inhibitorbinds to an active site of the ACK and the Michael acceptor moiety ofirreversible inhibitor gains access (in one embodiment the step ofbinding leads to a conformational change in the ACK, thus exposing thecysteine) or is otherwise exposed to the cysteine residue of the ACK; asa result a covalent bond is formed between the “S” of the cysteineresidue and the Michael acceptor of the irreversible inhibitor.Consequently, the binding site moiety of the irreversible inhibitorremains bound or otherwise blocks the active site of the ACK.

In one embodiment, the ACK is Btk, a homolog of Btk or a tyrosine kinasehaving a cysteine residue in an amino acid sequence position that ishomologous to the amino acid sequence position of cysteine 481 in Btk.In some embodiments, the ACK is HER4. Inhibitor compounds describedherein include a Michael acceptor moiety, a binding site moiety and alinker that links the binding site moiety and the Michael acceptormoiety (and in some embodiments, the structure of the linker provides aconformation, or otherwise directs the Michael acceptor moiety, so as toimprove the selectivity of the irreversible inhibitor for a particularACK).

In some embodiments, the ACK inhibitor is a compound of Formula (A):

wherein

-   -   A is independently selected from N or CR₅;    -   R₁ is H, L₂-(substituted or unsubstituted alkyl),        L₂-(substituted or unsubstituted cycloalkyl), L₂-(substituted or        unsubstituted alkenyl), L₂-(substituted or unsubstituted        cycloalkenyl), L₂-(substituted or unsubstituted heterocycle),        L₂-(substituted or unsubstituted heteroaryl), or L₂-(substituted        or unsubstituted aryl), where L₂ is a bond, O, S, —S(═O),        —S(═O)₂, C(═O), -(substituted or unsubstituted C₁-C₆ alkyl), or        -(substituted or unsubstituted C₂-C₆ alkenyl);    -   R₂ and R₃ are independently selected from H, lower alkyl and        substituted lower alkyl;    -   R₄ is L₃-X-L₄-G, wherein,        -   L₃ is optional, and when present is a bond, optionally            substituted or unsubstituted alkyl, optionally substituted            or unsubstituted cycloalkyl, optionally substituted or            unsubstituted alkenyl, optionally substituted or            unsubstituted alkynyl;        -   X is optional, and when present is a bond, O, —C(═O), S,            —S(═O), —S(═O)₂, —NH, —NR₉, —NHC(O), —C(O)NH, —NR₉C(O),            —C(O)NR₉, —S(═O)₂NH, —NHS(═O)₂, —S(═O)₂NR₉—, —NR₉S(═O)₂,            —OC(O)NH—, —NHC(O)O—, —OC(O)NR₉—, —NR₉C(O)O—, —CH═NO—,            —ON═CH—, —NR₁₀C(O)NR₁₀—, heteroaryl, aryl,            —NR₁₀C(═NR₁₁)NR₁₀—, —NR₁₀C(═NR₁₁)—, —C(═NR₁₁)NR₁₀—,            —OC(═NR₁₁)—, or —C(═NR₁₁)O—;        -   L₄ is optional, and when present is a bond, substituted or            unsubstituted alkyl, substituted or unsubstituted            cycloalkyl, substituted or unsubstituted alkenyl,            substituted or unsubstituted alkynyl, substituted or            unsubstituted aryl, substituted or unsubstituted heteroaryl,            substituted or unsubstituted heterocycle;        -   or L₃, X and L₄ taken together form a nitrogen containing            heterocyclic ring;        -   G is

-   -   -    wherein,            -   R₆, R₇ and R₈ are independently selected from among H,                lower alkyl or substituted lower alkyl, lower                heteroalkyl or substituted lower heteroalkyl,                substituted or unsubstituted lower cycloalkyl, and                substituted or unsubstituted lower heterocycloalkyl;

    -   R₅ is H, halogen, -L₆-(substituted or unsubstituted C₁-C₃        alkyl), -L₆-(substituted or unsubstituted C₂-C₄ alkenyl),        -L₆-(substituted or unsubstituted heteroaryl), or        -L₆-(substituted or unsubstituted aryl), wherein L₆ is a bond,        O, S, —S(═O), S(═O)₂, NH, C(O), —NHC(O)O, —OC(O)NH, —NHC(O), or        —C(O)NH;

    -   each R₉ is independently selected from among H, substituted or        unsubstituted lower alkyl, and substituted or unsubstituted        lower cycloalkyl;

    -   each R₁₀ is independently H, substituted or unsubstituted lower        alkyl, or substituted or unsubstituted lower cycloalkyl; or

    -   two R₁₀ groups can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or

    -   R₁₀ and R₁₁ can together form a 5-, 6-, 7-, or 8-membered        heterocyclic ring; or

    -   each R₁₁ is independently selected from H or alkyl; and        pharmaceutically active metabolites, pharmaceutically acceptable        solvates, pharmaceutically acceptable salts, or pharmaceutically        acceptable prodrugs thereof.

In one embodiment, the compound of Formula (A) has the structure:

wherein:

-   A is N;-   R₂ and R₃ are each H;-   R₁ is phenyl-O-phenyl or phenyl-S-phenyl; and-   R₄ is L₃-X-L₄-G, wherein,    -   L₃ is optional, and when present is a bond, optionally        substituted or unsubstituted alkyl, optionally substituted or        unsubstituted cycloalkyl, optionally substituted or        unsubstituted alkenyl, optionally substituted or unsubstituted        alkynyl;    -   X is optional, and when present is a bond, O, —C(═O), S, —S(═O),        —S(═O)₂, —NH, —NR₉, —NHC(O), —C(O)NH, —NR₉C(O), —C(O)NR₉,        —S(═O)₂NH, —NHS(═O)₂, —S(═O)₂NR₉—, —NR₉S(═O)₂, —OC(O)NH—,        —NHC(O)O—, —OC(O)NR₉—, —NR₉C(O)O—, —CH═NO—, —ON═CH—,        —NR₁₀C(O)NR₁₀—, heteroaryl, aryl, —NR₁₀C(═NR₁₁)NR₁₀—,        —NR₁₀C(═NR₁₁)—, —C(═NR₁₁)NR₁₀—, —OC(═NR₁₁)—, or —C(═NR₁₁)O—;    -   L₄ is optional, and when present is a bond, substituted or        unsubstituted alkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted alkenyl, substituted or        unsubstituted alkynyl, substituted or unsubstituted aryl,        substituted or unsubstituted heteroaryl, substituted or        unsubstituted heterocycle;    -   or L₃, X and L₄ taken together form a nitrogen containing        heterocyclic ring;    -   G is

wherein, R₆, R₇ and R₈ are independently selected from among H, loweralkyl or substituted lower alkyl, lower heteroalkyl or substituted lowerheteroalkyl, substituted or unsubstituted lower cycloalkyl, andsubstituted or unsubstituted lower heterocycloalkyl.

In some embodiments, the ACK inhibitor is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib)

In some embodiments, the ACK inhibitor is PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13.

In some embodiments, the ACK inhibitor is4-(tert-butyl)-N-(2-methyl-3-(4-methyl-6-((4-(morpholine-4-carbonyl)phenyl)amino)-5-oxo-4,5-dihydropyrazin-2-yl)phenyl)benzamide(CGI-1746);7-benzyl-1-(3-(piperidin-1-yl)propyl)-2-(4-(pyridin-4-yl)phenyl)-1H-imidazo[4,5-g]quinoxalin-6(5H)-one(CTA-056);(R)—N-(3-(6-(4-(1,4-dimethyl-3-oxopiperazin-2-yl)phenylamino)-4-methyl-5-oxo-4,5-dihydropyrazin-2-yl)-2-methylphenyl)-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide(GDC-0834);6-cyclopropyl-8-fluoro-2-(2-hydroxymethyl-3-{1-methyl-5-[5-(4-methyl-piperazin-1-yl)-pyridin-2-ylamino]-6-oxo-1,6-dihydro-pyridin-3-yl}-phenyl)-2H-isoquinolin-1-one(RN-486);N-[5-[5-(4-acetylpiperazine-1-carbonyl)-4-methoxy-2-methylphenyl]sulfanyl-1,3-thiazol-2-yl]-4-[(3,3-dimethylbutan-2-ylamino)methyl]benzamide(BMS-509744, HY-11092); orN-(5-((5-(4-Acetylpiperazine-1-carbonyl)-4-methoxy-2-methylphenyl)thio)thiazol-2-yl)-4-(((3-methylbutan-2-yl)amino)methyl)benzamide(HY11066).

In some embodiments, the ACK inhibitor is:

Combination Therapies

Described herein are methods of treating HER2 amplified breast cancer inan individual in need thereof comprising co-administering to theindividual a composition comprising a therapeutically-effective amountof an ACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) and an additional therapeutic agent. Further described hereinare methods of treating HER2 amplified cancer in an individual in needthereof comprising co-administering to the individual a compositioncomprising a therapeutically-effective amount of an ACK inhibitorcompound (e.g., a BTK inhibitor, such as for example ibrutinib) and anadditional therapeutic agent. In some embodiments, the ACK inhibitorcompound is a BTK inhibitor. In some embodiments, the BTK compound isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is selectedfrom among ibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101(Avila Therapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

In some embodiments, the additional therapeutic agent is an anticanceragent. In some embodiments, the additional therapeutic agent is achemotherapeutic agent. Classes of chemotherapeutic agents include, butare not limited to: alkylating agents, antimetabolites, spindle poisonplant alkaloids, cytotoxic/antitumor antibiotics, topoisomeraseinhibitors, antibodies, photosensitizers, and kinase inhibitors.Chemotherapeutic agents for administration with ibrutinib includecompounds used in “targeted therapy” and conventional chemotherapy.

In some embodiments, described herein are methods of treating HER2amplified breast cancer in an individual in need thereof comprisingco-administering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound and ananticancer agent. In some embodiments, also described herein are methodsof treating HER2 amplified cancer in an individual in need thereofcomprising co-administering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound and ananticancer agent.

In some embodiments, described herein are methods of treating HER2amplified breast cancer in an individual in need thereof comprisingco-administering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound and achemotherapeutic agent. In some embodiments, also described herein aremethods of treating HER2 amplified cancer in an individual in needthereof comprising co-administering to the individual a compositioncomprising a therapeutically-effective amount of an ACK inhibitorcompound and a chemotherapeutic agent. In some embodiments, the ACKinhibitor compound is selected from among ibrutinib (PCI-32765),PCI-45292, PCI-45466, AVL-101/CC-101 (Avila Therapeutics/CelgeneCorporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291(Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

In some embodiments, described herein are methods of treating HER2amplified breast cancer in an individual in need thereof comprisingco-administering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound andibrutinib. In some embodiments, also described herein are methods oftreating HER2 amplified cancer in an individual in need thereofcomprising co-administering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound andibrutinib.

Examples of chemotherapeutic agents for administration with ibrutinibfor treatment of a HER2-amplified cancer include, but are not limitedto: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®,Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8),gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer),cisplatin (cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1),carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-MyersSquibb Oncology, Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech),temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN@), Akti-1/2,HPPD, rapamycin, and any combinations thereof.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor (e.g. a BTK inhibitor, such as for exampleibrutinib) for treatment of a HER2-amplified cancer selected from among:oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, MillenniumPharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®,Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (MEKinhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244,Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, SemaforePharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3Kinhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant(FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin(sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmith Kline), lonafarnib (SARASAR™ SCH 66336, Schering Plough),sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®,AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib(ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free),albumin-engineered nanoparticle formulations of paclitaxel (AmericanPharmaceutical Partners, Schaumberg, Ill.), vandetanib (rINN, ZD6474,ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen),temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline),canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide(CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (T-2 toxin, verracurin A,roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine;mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C);cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine(NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above and any combinations thereof.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor (e.g. a BTK inhibitor, such as for exampleibrutinib) for treatment of a HER2-amplified cancer is selected fromamong (i) anti-hormonal agents that act to regulate or inhibit hormoneaction on tumors such as antiestrogens and selective estrogen receptormodulators (SERMs), including, for example, tamoxifen (NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above, and any combinationsthereof.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor (e.g. a BTK inhibitor, such as for exampleibrutinib) for treatment of a HER2-amplified cancer selected from amongtherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibodydrug-conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth) and anycombinations thereof.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor (e.g. a BTK inhibitor, such as for exampleibrutinib) for treatment of a HER2-amplified cancer is a humanizedmonoclonal antibody selected from among: alemtuzumab, apolizumab,aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumabmertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol,cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab,epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin,inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab,mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab,nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab,pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab,ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab,rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab,tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab,tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin,tucusituzumab, umavizumab, urtoxazumab, and visilizumab. In someembodiments, the additional therapeutic agent is selected fromerlotinib, docetaxel, 5-FU, gemcitabine, PD-0325901, cisplatin,carboplatin, paclitaxel, bevacizumab, trastuzumab, pertuzumab,temozolomide, tamoxifen, doxorubicin, Akti-1/2, HPPD, rapamycin,lapatinib, and any combinations thereof.

In some embodiments, the additional chemotherapeutic agent foradministration with an AKT inhibitor (e.g. a BTK inhibitor, such as forexample ibrutinib) for treatment of a HER2-amplified cancer is selectedfrom among an antibody, a B cell receptor pathway inhibitor, a T cellreceptor inhibitor, a PI3K inhibitor, an IAP inhibitor, an mTORinhibitor, a radioimmunotherapeutic, a DNA damaging agent, a proteasomeinhibitor, a histone deacetylase (HDCA) inhibitor, a protein kinaseinhibitor, an IRAK inhibitor, a hedgehog inhibitor, an Hsp90 inhibitor,a telomerase inhibitor, a Jak1/2 inhibitor (e.g., ruxolitinib,baricitinib, CYT387, lestauritinib, pacritinib, TG101348, SAR302503,tofacitinib (Xeljanz), etanercept (Enbrel), GLPG0634, R256), a proteaseinhibitor, a PKC inhibitor, a PARP inhibitor, a proteosome inhibitor, aCYP3A4 inhibitor, an AKT inhibitor, an Erk inhibitor, an alkylatingagent, an anti metabolite, a plant alkaloid, a terpenoid, a cytotoxin, atopoisomerase inhibitor, or a combination thereof. In some embodiments,the B cell receptor pathway inhibitor is a CD79A inhibitor, a CD79Binhibitor, a CD19 inhibitor, a Lyn inhibitor, a Syk inhibitor, a PI3Kinhibitor, a Blnk inhibitor, a PLCγ inhibitor, a PKCβ inhibitor, a CD22inhibitor, a Bcl-2 inhibitor, an IRAK 1/4 inhibitor, a microtubuleinhibitor, a Topo II inhibitor, anti TWEAK, anti-IL17 bispecificantibody, a CK2 inhibitor, anaplastic lymphoma kinase (ALK) and c-Metinhibitors, a T cell receptor inhibitor is Muromonab-CD3, demethylaseenzyme inhibitors such as demethylase, HDM, LSDI and KDM, fatty acidsynthase inhibitors such as spirocyclic piperidine derivatives,glucocorticosteriod receptor agonist, fusion anti-CD 19-cytotoxic agentconjugate, antimetabolite, p70S6K inhibitor, immune modulators, AKT/PKBinhibitor, procaspase-3 activator PAC-1, BRAF inhibitor, lactatedehydrogenase A (LDH-A) inhibitor, CCR2 inhibitor, CXCR4 inhibitor,chemokine receptor antagonists, DNA double stranded break repairinhibitors, NOR202, GA-101, TLR2 inhibitor, and any combinationsthereof.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer is ananti-HER2 therapeutic agent. In some embodiments, the additionaltherapeutic agent for administration with an ACK inhibitor for treatmentof a HER2-amplified cancer is a kinase inhibitor. In some embodiments,the additional therapeutic agent for administration with an ACKinhibitor for treatment of a HER2-amplified cancer is selected from thegroup consisting of: trastuzumab, trastuzumab emtansine, pertuzumab,lapatinib, and MM-111 (Merrimack Pharmaceuticals). In some embodiments,the additional therapeutic agent for administration with an ACKinhibitor for treatment of a HER2-amplified cancer is trastuzumab. Insome embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer istrastuzumab emtansine. In some embodiments, the additional therapeuticagent for administration with an ACK inhibitor for treatment of aHER2-amplified cancer is lapatinib. In some embodiments, the additionaltherapeutic agent for administration with an ACK inhibitor for treatmentof a HER2-amplified cancer is pertuzumab. In some embodiments, theadditional therapeutic agent for administration with an ACK inhibitorfor treatment of a HER2-amplified cancer is MM-111. In some embodiments,the ACK inhibitor is a BTK inhibitor. In some embodiments, the ACKinhibitor compound is selected from among ibrutinib (PCI-32765),PCI-45292, PCI-45466, AVL-101/CC-101 (Avila Therapeutics/CelgeneCorporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291(Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is an anti-HER2therapeutic agent. In some embodiments, the additional therapeutic agentfor administration with ibrutinib for treatment of a HER2-amplifiedcancer is a kinase inhibitor. In some embodiments, the additionaltherapeutic agent for administration with ibrutinib for treatment of aHER2-amplified cancer is selected from the group consisting of:trastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, and MM-111(Merrimack Pharmaceuticals). In some embodiments, the additionaltherapeutic agent for administration with ibrutinib for treatment of aHER2-amplified cancer is trastuzumab. In some embodiments, theadditional therapeutic agent for administration with ibrutinib fortreatment of a HER2-amplified cancer is trastuzumab emtansine. In someembodiments, the additional therapeutic agent for administration withibrutinib for treatment of a HER2-amplified cancer is lapatinib. In someembodiments, the additional therapeutic agent for administration withibrutinib for treatment of a HER2-amplified cancer is pertuzumab. Insome embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is MM-111.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer ispan-ErbB inhibitor. In some embodiments, the additional therapeuticagent for administration with an ACK inhibitor for treatment of aHER2-amplified cancer is selected from the group consisting of:afatinib, neratinib, and dacomitinib. In some embodiments, theadditional therapeutic agent for administration with an ACK inhibitorfor treatment of a HER2-amplified cancer is afatinib. In someembodiments, the additional therapeutic agent for administration with anACK inhibitor for treatment of a HER2-amplified cancer is neratinib. Insome embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer isdacomitinib. In some embodiments, the ACK inhibitor is a BTK inhibitor.In some embodiments, the ACK inhibitor compound is selected from amongibrutinib (PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutic s/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is pan-ErbBinhibitor. In some embodiments, the additional therapeutic agent foradministration with ibrutinib for treatment of a HER2-amplified canceris selected from the group consisting of: afatinib, neratinib, anddacomitinib. In some embodiments, the additional therapeutic agent foradministration with ibrutinib for treatment of a HER2-amplified canceris afatinib. In some embodiments, the additional therapeutic agent foradministration with ibrutinib for treatment of a HER2-amplified canceris neratinib. In some embodiments, the additional therapeutic agent foradministration with ibrutinib for treatment of a HER2-amplified canceris dacomitinib.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer is ananti-VEGF therapeutic agent. In some embodiments, the additionaltherapeutic agent for administration with an ACK inhibitor for treatmentof a HER2-amplified cancer is selected from the group consisting of:bevacizumab, ranibizumab, lapatinib, sunitinib, sorafenib, axitinib, andpazopanib. In some embodiments, the additional therapeutic agent foradministration with an ACK inhibitor for treatment of a HER2-amplifiedcancer is bevacizumab. In some embodiments, the additional therapeuticagent for administration with an ACK inhibitor for treatment of aHER2-amplified cancer is ranibizumab. In some embodiments, theadditional therapeutic agent for administration with an ACK inhibitorfor treatment of a HER2-amplified cancer is lapatinib. In someembodiments, the additional therapeutic agent for administration with anACK inhibitor for treatment of a HER2-amplified cancer is sunitinib. Insome embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer issorafenib. In some embodiments, the additional therapeutic agent foradministration with an ACK inhibitor for treatment of a HER2-amplifiedcancer is axitinib. In some embodiments, the additional therapeuticagent for administration with an ACK inhibitor for treatment of aHER2-amplified cancer is pazopanib. In some embodiments, the ACKinhibitor is a BTK inhibitor. In some embodiments, the ACK inhibitorcompound is selected from among ibrutinib (PCI-32765), PCI-45292,PCI-45466, AVL-101/CC-101 (Avila Therapeutic s/Celgene Corporation),AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292(Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is an anti-VEGFtherapeutic agent. In some embodiments, the additional therapeutic agentfor administration with ibrutinib for treatment of a HER2-amplifiedcancer is selected from the group consisting of: bevacizumab,ranibizumab, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib.In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is bevacizumab.In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is ranibizumab.In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is lapatinib. Insome embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is sunitinib. Insome embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is sorafenib. Insome embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is axitinib. Insome embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is pazopanib.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer isselected from the group consisting of: temsirolimus, paclitaxel,ASLAN001 (also, ARRY-543, ASLAN Pharmaceuticals), vorinostat,doxorubicin, cyclophosphamide, cisplatin, docetaxel, and dasatinib. Insome embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer isdocetaxel. In some embodiments, the additional therapeutic agent foradministration with an ACK inhibitor for treatment of a HER2-amplifiedcancer is paclitaxal. In some embodiments, the additional therapeuticagent for administration with an ACK inhibitor for treatment of aHER2-amplified cancer is doxorubicin. In some embodiments, the ACKinhibitor is a BTK inhibitor. In some embodiments, the ACK inhibitorcompound is selected from among ibrutinib (PCI-32765), PCI-45292,PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292(Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is selected fromthe group consisting of: temsirolimus, paclitaxel, ASLAN001 (also,ARRY-543, ASLAN Pharmaceuticals), vorinostat, doxorubicin,cyclophosphamide, cisplatin, docetaxel, and dasatinib. In someembodiments, the additional therapeutic agent for administration withibrutinib for treatment of a HER2-amplified cancer is docetaxel. In someembodiments, the additional therapeutic agent for administration withibrutinib for treatment of a HER2-amplified cancer is paclitaxal. Insome embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is doxorubicin.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer istrastuzumab and docetaxel. In some embodiments, the ACK inhibitor is aBTK inhibitor. In some embodiments, the ACK inhibitor compound isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib). In some embodiments, the additionaltherapeutic agent for administration with ibrutinib for treatment of aHER2-amplified cancer is trastuzumab and docetaxel.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer ispertuzumab and docetaxel. In some embodiments, the ACK inhibitor is aBTK inhibitor. In some embodiments, the ACK inhibitor compound isselected from among ibrutinib (PCI-32765), PCI-45292, PCI-45466,AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation), AVL-263/CC-263(Avila Therapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib). In some embodiments, the additionaltherapeutic agent for administration with ibrutinib for treatment of aHER2-amplified cancer is pertuzumab and docetaxel.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer isdoxorubicin, cyclophosphamide and paclitaxal. In some embodiments, theACK inhibitor is a BTK inhibitor. In some embodiments, the ACK inhibitorcompound is selected from among ibrutinib (PCI-32765), PCI-45292,PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292(Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the additional therapeutic agent foradministration with ibrutinib for treatment of a HER2-amplified canceris doxorubicin, cyclophosphamide and paclitaxal.

In some embodiments, the additional therapeutic agent for administrationwith an ACK inhibitor for treatment of a HER2-amplified cancer isdoxorubicin, cyclophosphamide and 5-FU. In some embodiments, the ACKinhibitor is a BTK inhibitor. In some embodiments, the ACK inhibitorcompound is selected from among ibrutinib (PCI-32765), PCI-45292,PCI-45466, AVL-101/CC-101 (Avila Therapeutics/Celgene Corporation),AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation), AVL-292/CC-292(Avila Therapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the additional therapeutic agent foradministration with ibrutinib for treatment of a HER2-amplified canceris doxorubicin, cyclophosphamide and 5-FU.

In some embodiments, the additional therapeutic agent for administrationwith ibrutinib for treatment of a HER2-amplified cancer is a histonedeacetylase (HDAC) inhibitor. In some embodiments, the HDAC inhibitor is3-[(dimethylamino)methyl]-N-{2-[4-(hydroxycarbamoyl)phenoxy]ethyl}-1-benzofuran-2-carboxamide(i.e. PCI-24781/abexinostat).

As described elsewhere herein, in some embodiments, the HER2-amplifiedcancer is selected from the group consisting of: breast, colon,endometrial, cervical, urothelial, lung (including, non-small cell lungcancer), ovarian, gastric, gastroesophageal junction (GEJ), head andneck, biliary tract, prostate, and pancreatic cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified breast cancer.In some embodiments, the HER2-amplified cancer is HER2-amplified coloncancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedendometrial cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified cervical cancer. In some embodiments, the HER2-amplifiedcancer is HER2-amplified urothelial cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified lung cancer. In someembodiments, the HER2-amplified lung cancer is HER2-amplified non-smallcell lung cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified ovarian cancer. In some embodiments, the HER2-amplifiedcancer is HER2-amplified gastric cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified gastroesophageal junction (GEJ)cancer. In some embodiments, the HER2-amplified cancer is HER2-amplifiedhead and neck cancer. In some embodiments, the HER2-amplified cancer isHER2-amplified biliary tract cancer. In some embodiments, theHER2-amplified cancer is HER2-amplified prostate cancer. In someembodiments, the HER2-amplified cancer is HER2-amplified pancreaticcancer. In some embodiments, the HER2-amplified cancer is metastatic. Insome embodiments, the HER2-amplified cancer has metastasized to thebrain. In some embodiments, the HER2-amplified cancer has a HER2:CEP17ratio >4.0. In some embodiments, the HER2-amplified cancer has aHER2:CEP17 ratio of 2.2-4.0. In some embodiments, the HER2-amplifiedcancer is graded 3+ using IHC. In some embodiments, the HER2-amplifiedcancer is refractory to treatment. In some embodiments, the treatment towhich the HER2-amplified cancer is refractory is selected from:trastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. Insome embodiments, the treatment to which the HER2-amplified cancer isrefractory is trastuzumab. In some embodiments, the HER2-amplifiedcancer is recurrent.

When an additional agent is co-administered with an ACK inhibitor, theadditional agent and the ACK inhibitor do not have to be administered inthe same pharmaceutical composition, and are optionally, because ofdifferent physical and chemical characteristics, administered bydifferent routes. The initial administration is made, for example,according to established protocols, and then, based upon the observedeffects, the dosage, modes of administration and times of administrationare modified.

By way of example only, if a side effect experienced by an individualupon receiving an ACK inhibitor is nausea, then it is appropriate toadminister an anti-emetic agent in combination with the ACK inhibitor.

Or, by way of example only, the therapeutic effectiveness of an ACKinhibitor described herein is enhanced by administration of an adjuvant(i.e., by itself the adjuvant has minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the patient is enhanced). Or, by way of example only, thebenefit experienced by an individual is increased by administering anACK inhibitor described herein with another therapeutic agent (whichalso includes a therapeutic regimen) that also has therapeutic benefit.In any case, regardless of the disease, disorder being treated, theoverall benefit experienced by the patient is in some embodiments simplyadditive of the two therapeutic agents or in other embodiments, thepatient experiences a synergistic benefit.

The particular choice of compounds used will depend upon the diagnosisof the attending physicians and their judgment of the condition of thepatient and the appropriate treatment protocol. The compounds areoptionally administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially,depending upon the nature of the disorder, the condition of the patient,and the actual choice of compounds used. The determination of the orderof administration, and the number of repetitions of administration ofeach therapeutic agent during a treatment protocol, is based on anevaluation of the disease being treated and the condition of thepatient.

In some embodiments, therapeutically-effective dosages vary when thedrugs are used in treatment combinations. Methods for experimentallydetermining therapeutically-effective dosages of drugs and other agentsfor use in combination treatment regimens are described in theliterature. For example, the use of metronomic dosing, i.e., providingmore frequent, lower doses in order to minimize toxic side effects, hasbeen described extensively in the literature Combination treatmentfurther includes periodic treatments that start and stop at varioustimes to assist with the clinical management of the patient.

For combination therapies described herein, dosages of theco-administered compounds will of course vary depending on the type ofco-drug employed, on the specific drug employed, on the disorder beingtreated and so forth. In addition, when co-administered with anadditional therapeutic agent, an ACK inhibitor described herein isadministered either simultaneously with the additional therapeuticagent, or sequentially. If administered sequentially, the attendingphysician will decide on the appropriate sequence of administeringprotein in combination with the biologically active agent(s).

If the additional therapeutic agent and the ACK inhibitor areadministered simultaneously, the multiple therapeutic agents areoptionally provided in a single, unified form, or in multiple forms (byway of example only, either as a single pill or as two separate pills).In some embodiments, one of the therapeutic agents is given in multipledoses, or both are given as multiple doses. If not simultaneous, thetiming between the multiple doses is from about more than zero weeks toless than about four weeks. In addition, the combination methods,compositions and formulations are not to be limited to the use of onlytwo agents; the use of multiple therapeutic combinations is alsoenvisioned.

It is understood that the dosage regimen to treat, prevent, orameliorate the condition(s) for which relief is sought, can be modifiedin accordance with a variety of factors. These factors include thedisorder from which the subject suffers, as well as the age, weight,sex, diet, and medical condition of the subject. Thus, the dosageregimen actually employed can vary widely and therefore can deviate fromthe dosage regimens set forth herein.

In some embodiments, the pharmaceutical agents which make up thecombination therapy disclosed herein are administered in a combineddosage form, or in separate dosage forms intended for substantiallysimultaneous administration. In some embodiments, the pharmaceuticalagents that make up the combination therapy are administeredsequentially, with either therapeutic compound being administered by aregimen calling for two-step administration. In some embodiments, thetwo-step administration regimen calls for sequential administration ofthe active agents or spaced-apart administration of the separate activeagents. The time period between the multiple administration steps rangesfrom a few minutes to several hours, depending upon the properties ofeach pharmaceutical agent, such as potency, solubility, bioavailability,plasma half-life and kinetic profile of the pharmaceutical agent. Insome embodiments, circadian variation of the target moleculeconcentration determines the optimal dose interval.

In some embodiments, the ACK inhibitor compound and the additionaltherapeutic agent are administered in a unified dosage form. In someembodiments, the ACK inhibitor compound and the additional therapeuticagent are administered in separate dosage forms. In some embodiments,the ACK inhibitor compound and the additional therapeutic agent areadministered simultaneously or sequentially.

Pharmaceutical Compositions/Formulations

Disclosed herein, in certain embodiments, are compositions comprising atherapeutically effective amount of an ACK inhibitor compound, and apharmaceutically acceptable excipient. In some embodiments, the ACKinhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) is a compound of Formula (A). In some embodiments, the ACKinhibitor compound is selected from among ibrutinib (PCI-32765),PCI-45292, PCI-45466, AVL-101/CC-101 (Avila Therapeutics/CelgeneCorporation), AVL-263/CC-263 (Avila Therapeutics/Celgene Corporation),AVL-292/CC-292 (Avila Therapeutics/Celgene Corporation), AVL-291/CC-291(Avila Therapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib).

Pharmaceutical compositions of ACK inhibitor compound (e.g., a BTKinhibitor, such as for example ibrutinib) are formulated in aconventional manner using one or more physiologically acceptablecarriers including excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. A summary of pharmaceutical compositionsdescribed herein is found, for example, in Remington: The Science andPractice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack PublishingCompany, 1995); Hoover, John E., Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980;and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins 1999).

A pharmaceutical composition, as used herein, refers to a mixture of anACK inhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) with other chemical components, such as carriers,stabilizers, diluents, dispersing agents, suspending agents, thickeningagents, and/or excipients.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions may also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

The term “pharmaceutical combination” as used herein, means a productthat results from the mixing or combining of more than one activeingredient and includes both fixed and non-fixed combinations of theactive ingredients. The term “fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, are bothadministered to a patient simultaneously in the form of a single entityor dosage. The term “non-fixed combination” means that the activeingredients, e.g. a compound described herein and a co-agent, areadministered to a patient as separate entities either simultaneously,concurrently or sequentially with no specific intervening time limits,wherein such administration provides effective levels of the twocompounds in the body of the patient. The latter also applies tococktail therapy, e.g. the administration of three or more activeingredients.

Pharmaceutical compositions are optionally manufactured in aconventional manner, such as, by way of example only, by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or compression processes.

The pharmaceutical formulations described herein are administered by anysuitable administration route, including but not limited to, oral,parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal,buccal, topical, rectal, or transdermal administration routes.

The pharmaceutical compositions described herein are formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions andthe like, for oral ingestion by an individual to be treated, solid oraldosage forms, aerosols, controlled release formulations, fast meltformulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations. In some embodiments, thecompositions are formulated into capsules. In some embodiments, thecompositions are formulated into solutions (for example, for IVadministration).

The pharmaceutical solid dosage forms described herein optionallyinclude a compound described herein and one or more pharmaceuticallyacceptable additives such as a compatible carrier, binder, fillingagent, suspending agent, flavoring agent, sweetening agent,disintegrating agent, dispersing agent, surfactant, lubricant, colorant,diluent, solubilizer, moistening agent, plasticizer, stabilizer,penetration enhancer, wetting agent, anti-foaming agent, antioxidant,preservative, or one or more combination thereof.

In still other aspects, using standard coating procedures, such as thosedescribed in Remington's Pharmaceutical Sciences, 20th Edition (2000), afilm coating is provided around the compositions. In some embodiments,the compositions are formulated into particles (for example foradministration by capsule) and some or all of the particles are coated.In some embodiments, the compositions are formulated into particles (forexample for administration by capsule) and some or all of the particlesare microencapsulated. In some embodiments, the compositions areformulated into particles (for example for administration by capsule)and some or all of the particles are not microencapsulated and areuncoated.

In some embodiments, the pharmaceutical compositions are formulated suchthat the amount of the ACK inhibitor (e.g., a BTK inhibitor, such as forexample ibrutinib) in each unit dosage form is about 140 mg per day.

Dosage Forms

The compositions described herein can be formulated for administrationto a subject via any conventional means including, but not limited to,oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular),buccal, intranasal, rectal or transdermal administration routes. In someembodiments, the composition is formulated for administration in aseparate dosage form. In some embodiments, the composition is formulatedfor administration in a combined dosage form.

In some embodiments, the pharmaceutical compositions described herein,which include ibrutinib and/or an additional therapeutic agent can beformulated into any suitable dosage form, including but not limited to,aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries,suspensions and the like, for oral ingestion by a patient to be treated,solid oral dosage forms, aerosols, controlled release formulations, fastmelt formulations, effervescent formulations, lyophilized formulations,tablets, powders, pills, dragees, capsules, delayed releaseformulations, extended release formulations, pulsatile releaseformulations, multiparticulate formulations, and mixed immediate releaseand controlled release formulations.

Pharmaceutical preparations for oral use can be obtained by mixing oneor more solid excipient with one or more of the compounds describedherein, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include, forexample, fillers such as sugars, including lactose, sucrose, mannitol,or sorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents may be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

In some embodiments, the solid dosage forms disclosed herein may be inthe form of a tablet, (including a suspension tablet, a fast-melttablet, a bite-disintegration tablet, a rapid-disintegration tablet, aneffervescent tablet, or a caplet), a pill, a powder (including a sterilepackaged powder, a dispensable powder, or an effervescent powder) acapsule (including both soft or hard capsules, e.g., capsules made fromanimal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”),solid dispersion, solid solution, bioerodible dosage form, controlledrelease formulations, pulsatile release dosage forms, multiparticulatedosage forms, pellets, granules, or an aerosol. In other embodiments,the pharmaceutical formulation is in the form of a powder. In stillother embodiments, the pharmaceutical formulation is in the form of atablet, including but not limited to, a fast-melt tablet. Additionally,pharmaceutical formulations described herein may be administered as asingle capsule or in multiple capsule dosage form. In some embodiments,the pharmaceutical formulation is administered in two, or three, orfour, capsules or tablets.

In some embodiments, solid dosage forms, e.g., tablets, effervescenttablets, and capsules, are prepared by mixing particles of ibrutiniband/or an anticancer agent, with one or more pharmaceutical excipientsto form a bulk blend composition. When referring to these bulk blendcompositions as homogeneous, it is meant that the particles of ibrutiniband/or an additional therapeutic agent, are dispersed evenly throughoutthe composition so that the composition may be readily subdivided intoequally effective unit dosage forms, such as tablets, pills, andcapsules. The individual unit dosages may also include film coatings,which disintegrate upon oral ingestion or upon contact with diluent.These formulations can be manufactured by conventional pharmacologicaltechniques.

Conventional pharmacological techniques include, e.g., one or acombination of methods: (1) dry mixing, (2) direct compression, (3)milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6)fusion. See, e.g., Lachman et al., The Theory and Practice of IndustrialPharmacy (1986). Other methods include, e.g., spray drying, pan coating,melt granulation, granulation, fluidized bed spray drying or coating(e.g., wurster coating), tangential coating, top spraying, tableting,extruding and the like.

The pharmaceutical solid dosage forms described herein can include acompound described herein and one or more pharmaceutically acceptableadditives such as a compatible carrier, binder, filling agent,suspending agent, flavoring agent, sweetening agent, disintegratingagent, dispersing agent, surfactant, lubricant, colorant, diluent,solubilizer, moistening agent, plasticizer, stabilizer, penetrationenhancer, wetting agent, anti-foaming agent, antioxidant, preservative,or one or more combination thereof. In still other aspects, usingstandard coating procedures, such as those described in Remington'sPharmaceutical Sciences, 20th Edition (2000), a film coating is providedaround the formulation of ibrutinib and/or an anticancer agent. Inanother embodiment, some or all of the particles of ibrutinib and/or ananticancer agent, are not microencapsulated and are uncoated.

Suitable carriers for use in the solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Suitable filling agents for use in the solid dosage forms describedherein include, but are not limited to, lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, hydroxypropylmethycellulose(HPMC), hydroxypropylmethycellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose,xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethyleneglycol, and the like.

In order to release the compound of ibrutinib and/or an additionaltherapeutic agent, from a solid dosage form matrix as efficiently aspossible, disintegrants are often used in the formulation, especiallywhen the dosage forms are compressed with binder. Disintegrants helprupturing the dosage form matrix by swelling or capillary action whenmoisture is absorbed into the dosage form. Suitable disintegrants foruse in the solid dosage forms described herein include, but are notlimited to, natural starch such as corn starch or potato starch, apregelatinized starch such as National 1551 or Amijel®, or sodium starchglycolate such as Promogel® or Explotab®, a cellulose such as a woodproduct, methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101,Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, MingTia®, and Solka-Floc®, methylcellulose, croscarmellose, or across-linked cellulose, such as cross-linked sodiumcarboxymethylcellulose (Ac-Di-Sol®), cross-linkedcarboxymethylcellulose, or cross-linked croscarmellose, a cross-linkedstarch such as sodium starch glycolate, a cross-linked polymer such ascrospovidone, a cross-linked polyvinylpyrrolidone, alginate such asalginic acid or a salt of alginic acid such as sodium alginate, a claysuch as Veegum® HV (magnesium aluminum silicate), a gum such as agar,guar, locust bean, Karaya, pectin, or tragacanth, sodium starchglycolate, bentonite, a natural sponge, a surfactant, a resin such as acation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium laurylsulfate in combination starch, and the like.

Binders impart cohesiveness to solid oral dosage form formulations: forpowder filled capsule formulation, they aid in plug formation that canbe filled into soft or hard shell capsules and for tablet formulation,they ensure the tablet remaining intact after compression and helpassure blend uniformity prior to a compression or fill step. Materialssuitable for use as binders in the solid dosage forms described hereininclude, but are not limited to, carboxymethylcellulose, methylcellulose(e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USPPharmacoat-603, hydroxypropylmethylcellulose acetate stearate (AqoateHS-LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g.,Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystallinecellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesiumaluminum silicate, polysaccharide acids, bentonites, gelatin,polyvinylpyrrolidone/vinyl acetate copolymer, crospovidone, povidone,starch, pregelatinized starch, tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such asacacia, tragacanth, ghatti gum, mucilage of isapol husks, starch,polyvinylpyrrolidone (e.g., Povidone® CL, Kollidon® CL, Polyplasdone®XL-10, and Povidone® K-12), larch arabogalactan, Veegum®, polyethyleneglycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder. Formulators skilled in art can determine the binder level forthe formulations, but binder usage level of up to 70% in tabletformulations is common.

Suitable lubricants or glidants for use in the solid dosage formsdescribed herein include, but are not limited to, stearic acid, calciumhydroxide, talc, corn starch, sodium stearyl fumerate, alkali-metal andalkaline earth metal salts, such as aluminum, calcium, magnesium, zinc,stearic acid, sodium stearates, magnesium stearate, zinc stearate,waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol or a methoxypolyethylene glycolsuch as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol,sodium oleate, glyceryl behenate, glyceryl palmitostearate, glycerylbenzoate, magnesium or sodium lauryl sulfate, and the like.

Suitable diluents for use in the solid dosage forms described hereininclude, but are not limited to, sugars (including lactose, sucrose, anddextrose), polysaccharides (including dextrates and maltodextrin),polyols (including mannitol, xylitol, and sorbitol), cyclodextrins andthe like.

The term “non water-soluble diluent” represents compounds typically usedin the formulation of pharmaceuticals, such as calcium phosphate,calcium sulfate, starches, modified starches and microcrystallinecellulose, and microcellulose (e.g., having a density of about 0.45g/cm³, e.g. Avicel, powdered cellulose), and talc.

Suitable wetting agents for use in the solid dosage forms describedherein include, for example, oleic acid, glyceryl monostearate, sorbitanmonooleate, sorbitan monolaurate, triethanolamine oleate,polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodiumoleate, sodium lauryl sulfate, magnesium stearate, sodium docusate,triacetin, vitamin E TPGS and the like.

Suitable surfactants for use in the solid dosage forms described hereininclude, for example, sodium lauryl sulfate, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide, e.g., Pluronic® (BASF), and the like.

Suitable suspending agents for use in the solid dosage forms describedhere include, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Suitable antioxidants for use in the solid dosage forms described hereininclude, for example, e.g., butylated hydroxytoluene (BHT), sodiumascorbate, and tocopherol.

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included in solid dosageforms described herein. The amounts of such additives can be readilydetermined by one skilled in the art, according to the particularproperties desired.

In other embodiments, one or more layers of the pharmaceuticalformulation are plasticized. Illustratively, a plasticizer is generallya high boiling point solid or liquid. Suitable plasticizers can be addedfrom about 0.01% to about 50% by weight (w/w) of the coatingcomposition. Plasticizers include, but are not limited to, diethylphthalate, citrate esters, polyethylene glycol, glycerol, acetylatedglycerides, triacetin, polypropylene glycol, polyethylene glycol,triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, andcastor oil.

Compressed tablets are solid dosage forms prepared by compacting thebulk blend of the formulations described above. In various embodiments,compressed tablets which are designed to dissolve in the mouth willinclude one or more flavoring agents. In other embodiments, thecompressed tablets will include a film surrounding the final compressedtablet. In some embodiments, the film coating can provide a delayedrelease of ibrutinib or the second agent, from the formulation. In otherembodiments, the film coating aids in patient compliance (e.g., Opadry®coatings or sugar coating). Film coatings including Opadry® typicallyrange from about 1% to about 3% of the tablet weight. In otherembodiments, the compressed tablets include one or more excipients.

A capsule may be prepared, for example, by placing the bulk blend of theformulation of ibrutinib or an additional therapeutic agent, describedabove, inside of a capsule. In some embodiments, the formulations(non-aqueous suspensions and solutions) are placed in a soft gelatincapsule. In other embodiments, the formulations are placed in standardgelatin capsules or non-gelatin capsules such as capsules comprisingHPMC. In other embodiments, the formulation is placed in a sprinklecapsule, wherein the capsule may be swallowed whole or the capsule maybe opened and the contents sprinkled on food prior to eating. In someembodiments, the therapeutic dose is split into multiple (e.g., two,three, or four) capsules. In some embodiments, the entire dose of theformulation is delivered in a capsule form.

In various embodiments, the particles of ibrutinib and/or an additionaltherapeutic agent, and one or more excipients are dry blended andcompressed into a mass, such as a tablet, having a hardness sufficientto provide a pharmaceutical composition that substantially disintegrateswithin less than about 30 minutes, less than about 35 minutes, less thanabout 40 minutes, less than about 45 minutes, less than about 50minutes, less than about 55 minutes, or less than about 60 minutes,after oral administration, thereby releasing the formulation into thegastrointestinal fluid.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

Materials useful for the microencapsulation described herein includematerials compatible with ibrutinib and/or an additional therapeuticagent, which sufficiently isolate the compound of any of ibrutinib or anadditional therapeutic agent, from other non-compatible excipients.Materials compatible with compounds of any of ibrutinib or an additionaltherapeutic agent, are those that delay the release of the compounds ofany of ibrutinib or an additional therapeutic agent, in vivo.

Exemplary microencapsulation materials useful for delaying the releaseof the formulations including compounds described herein, include, butare not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel®or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC),hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC,Pharmacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, BenecelMP824, and Benecel MP843, methylcellulose polymers such as Methocel®-A,hydroxypropylmethylcellulose acetate stearate Aqoat (HF-LS, HF-LG,HF-MS) and Metolose®, Ethylcelluloses (EC) and mixtures thereof such asE461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinyl alcohol (PVA) such asOpadry AMB, hydroxyethylcelluloses such as Natrosol®,carboxymethylcelluloses and salts of carboxymethylcelluloses (CMC) suchas Aqualon®-CMC, polyvinyl alcohol and polyethylene glycol co-polymerssuch as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX),polyethylene glycols, modified food starch, acrylic polymers andmixtures of acrylic polymers with cellulose ethers such as Eudragit®EPO, Eudragit® L30D-55, Eudragit® FS 30D Eudragit® L100-55, Eudragit®L100, Eudragit® 5100, Eudragit® RD100, Eudragit® E100, Eudragit® L12.5,Eudragit® 512.5, Eudragit® NE30D, and Eudragit® NE 40D, celluloseacetate phthalate, sepifilms such as mixtures of HPMC and stearic acid,cyclodextrins, and mixtures of these materials.

In still other embodiments, plasticizers such as polyethylene glycols,e.g., PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800,stearic acid, propylene glycol, oleic acid, and triacetin areincorporated into the microencapsulation material. In other embodiments,the microencapsulating material useful for delaying the release of thepharmaceutical compositions is from the USP or the National Formulary(NF). In yet other embodiments, the microencapsulation material isKlucel. In still other embodiments, the microencapsulation material ismethocel.

Microencapsulated compounds of any of ibrutinib or an additionaltherapeutic agent may be formulated by methods known by one of ordinaryskill in the art. Such known methods include, e.g., spray dryingprocesses, spinning disk-solvent processes, hot melt processes, spraychilling methods, fluidized bed, electrostatic deposition, centrifugalextrusion, rotational suspension separation, polymerization atliquid-gas or solid-gas interface, pressure extrusion, or sprayingsolvent extraction bath. In addition to these, several chemicaltechniques, e.g., complex coacervation, solvent evaporation,polymer-polymer incompatibility, interfacial polymerization in liquidmedia, in situ polymerization, in-liquid drying, and desolvation inliquid media could also be used. Furthermore, other methods such asroller compaction, extrusion/spheronization, coacervation, ornanoparticle coating may also be used.

In one embodiment, the particles of compounds of any of ibrutinib or anadditional therapeutic agent are microencapsulated prior to beingformulated into one of the above forms. In still another embodiment,some or most of the particles are coated prior to being furtherformulated by using standard coating procedures, such as those describedin Remington's Pharmaceutical Sciences, 20th Edition (2000).

In other embodiments, the solid dosage formulations of the compounds ofany of ibrutinib and/or an additional therapeutic agent are plasticized(coated) with one or more layers. Illustratively, a plasticizer isgenerally a high boiling point solid or liquid. Suitable plasticizerscan be added from about 0.01% to about 50% by weight (w/w) of thecoating composition. Plasticizers include, but are not limited to,diethyl phthalate, citrate esters, polyethylene glycol, glycerol,acetylated glycerides, triacetin, polypropylene glycol, polyethyleneglycol, triethyl citrate, dibutyl sebacate, stearic acid, stearol,stearate, and castor oil.

In other embodiments, a powder including the formulations with acompound of any of ibrutinib and/or an anticancer agent, describedherein, may be formulated to include one or more pharmaceuticalexcipients and flavors. Such a powder may be prepared, for example, bymixing the formulation and optional pharmaceutical excipients to form abulk blend composition. Additional embodiments also include a suspendingagent and/or a wetting agent. This bulk blend is uniformly subdividedinto unit dosage packaging or multi-dosage packaging units.

In still other embodiments, effervescent powders are also prepared inaccordance with the present disclosure. Effervescent salts have beenused to disperse medicines in water for oral administration.Effervescent salts are granules or coarse powders containing a medicinalagent in a dry mixture, usually composed of sodium bicarbonate, citricacid and/or tartaric acid. When salts of the compositions describedherein are added to water, the acids and the base react to liberatecarbon dioxide gas, thereby causing “effervescence.” Examples ofeffervescent salts include, e.g., the following ingredients: sodiumbicarbonate or a mixture of sodium bicarbonate and sodium carbonate,citric acid and/or tartaric acid. Any acid-base combination that resultsin the liberation of carbon dioxide can be used in place of thecombination of sodium bicarbonate and citric and tartaric acids, as longas the ingredients were suitable for pharmaceutical use and result in apH of about 6.0 or higher.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine of the gastrointestinal tract. The enteric coated dosage formmay be a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components, which arethemselves coated or uncoated. The enteric coated oral dosage form mayalso be a capsule (coated or uncoated) containing pellets, beads orgranules of the solid carrier or the composition, which are themselvescoated or uncoated.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationin the intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments the method for delay of release is coating. Any coatingsshould be applied to a sufficient thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below about 5,but does dissolve at pH about 5 and above. It is expected that anyanionic polymer exhibiting a pH-dependent solubility profile can be usedas an enteric coating in the methods and compositions described hereinto achieve delivery to the lower gastrointestinal tract. In someembodiments the polymers described herein are anionic carboxylicpolymers. In other embodiments, the polymers and compatible mixturesthereof, and some of their properties, include, but are not limited to:

Shellac, also called purified lac, a refined product obtained from theresinous secretion of an insect. This coating dissolves in media of pH>7;

Acrylic polymers. The performance of acrylic polymers (primarily theirsolubility in biological fluids) can vary based on the degree and typeof substitution. Examples of suitable acrylic polymers includemethacrylic acid copolymers and ammonium methacrylate copolymers. TheEudragit series E, L, S, RL, RS and NE (Rohm Pharma) are available assolubilized in organic solvent, aqueous dispersion, or dry powders. TheEudragit series RL, NE, and RS are insoluble in the gastrointestinaltract but are permeable and are used primarily for colonic targeting.The Eudragit series E dissolve in the stomach. The Eudragit series L,L-30D and S are insoluble in stomach and dissolve in the intestine;

Cellulose Derivatives. Examples of suitable cellulose derivatives are:ethyl cellulose; reaction mixtures of partial acetate esters ofcellulose with phthalic anhydride. The performance can vary based on thedegree and type of substitution. Cellulose acetate phthalate (CAP)dissolves in pH >6. Aquateric (FMC) is an aqueous based system and is aspray dried CAP psuedolatex with particles <1 μm. Other components inAquateric can include pluronics, Tweens, and acetylated monoglycerides.Other suitable cellulose derivatives include: cellulose acetatetrimellitate (Eastman); methylcellulose (Pharmacoat, Methocel);hydroxypropylmethyl cellulose phthalate (HPMCP); hydroxypropylmethylcellulose succinate (HPMCS); and hydroxypropylmethylcellulose acetatesuccinate (e.g., AQOAT (Shin Etsu)). The performance can vary based onthe degree and type of substitution. For example, HPMCP such as, HP-50,HP-55, HP-555, HP-55F grades are suitable. The performance can varybased on the degree and type of substitution. For example, suitablegrades of hydroxypropylmethylcellulose acetate succinate include, butare not limited to, AS-LG (LF), which dissolves at pH 5, AS-MG (MF),which dissolves at pH 5.5, and AS-HG (HF), which dissolves at higher pH.These polymers are offered as granules, or as fine powders for aqueousdispersions; Poly Vinyl Acetate Phthalate (PVAP). PVAP dissolves inpH >5, and it is much less permeable to water vapor and gastric fluids.

In some embodiments, the coating can, and usually does, contain aplasticizer and possibly other coating excipients such as colorants,talc, and/or magnesium stearate, which are well known in the art.Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin(glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, anionic carboxylic acrylicpolymers usually will contain 10-25% by weight of a plasticizer,especially dibutyl phthalate, polyethylene glycol, triethyl citrate andtriacetin. Conventional coating techniques such as spray or pan coatingare employed to apply coatings. The coating thickness must be sufficientto ensure that the oral dosage form remains intact until the desiredsite of topical delivery in the intestinal tract is reached.

Colorants, detackifiers, surfactants, antifoaming agents, lubricants(e.g., carnuba wax or PEG) may be added to the coatings besidesplasticizers to solubilize or disperse the coating material, and toimprove coating performance and the coated product.

In other embodiments, the formulations described herein, which includeibrutinib and/or an additional therapeutic agent, are delivered using apulsatile dosage form. A pulsatile dosage form is capable of providingone or more immediate release pulses at predetermined time points aftera controlled lag time or at specific sites. Many other types ofcontrolled release systems known to those of ordinary skill in the artand are suitable for use with the formulations described herein.Examples of such delivery systems include, e.g., polymer-based systems,such as polylactic and polyglycolic acid, plyanhydrides andpolycaprolactone; porous matrices, nonpolymer-based systems that arelipids, including sterols, such as cholesterol, cholesterol esters andfatty acids, or neutral fats, such as mono-, di- and triglycerides;hydrogel release systems; silastic systems; peptide-based systems; waxcoatings, bioerodible dosage forms, compressed tablets usingconventional binders and the like. See, e.g., Liberman et al.,Pharmaceutical Dosage Forms, 2 Ed., Vol. 1, pp. 209-214 (1990); Singh etal., Encyclopedia of Pharmaceutical Technology, 2^(nd) Ed., pp. 751-753(2002); U.S. Pat. Nos. 4,327,725, 4,624,848, 4,968,509, 5,461,140,5,456,923, 5,516,527, 5,622,721, 5,686,105, 5,700,410, 5,977,175,6,465,014 and 6,932,983.

In some embodiments, pharmaceutical formulations are provided thatinclude particles of ibrutinib and/or an additional therapeutic agent,described herein and at least one dispersing agent or suspending agentfor oral administration to a subject. The formulations may be a powderand/or granules for suspension, and upon admixture with water, asubstantially uniform suspension is obtained.

Liquid formulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2^(nd) Ed., pp. 754-757(2002). In addition the liquid dosage forms may include additives, suchas: (a) disintegrating agents; (b) dispersing agents; (c) wettingagents; (d) at least one preservative, (e) viscosity enhancing agents,(f) at least one sweetening agent, and (g) at least one flavoring agent.In some embodiments, the aqueous dispersions can further include acrystalline inhibitor.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regard to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

Examples of disintegrating agents for use in the aqueous suspensions anddispersions include, but are not limited to, a starch, e.g., a naturalstarch such as corn starch or potato starch, a pregelatinized starchsuch as National 1551 or Amijel®, or sodium starch glycolate such asPromogel® or Explotab®; a cellulose such as a wood product,methylcrystalline cellulose, e.g., Avicel®, Avicel® PH101, Avicel®PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia®, andSolka-Floc®, methylcellulose, croscarmellose, or a cross-linkedcellulose, such as cross-linked sodium carboxymethylcellulose(Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linkedcroscarmellose; a cross-linked starch such as sodium starch glycolate; across-linked polymer such as crospovidone; a cross-linkedpolyvinylpyrrolidone; alginate such as alginic acid or a salt of alginicacid such as sodium alginate; a clay such as Veegum® HV (magnesiumaluminum silicate); a gum such as agar, guar, locust bean, Karaya,pectin, or tragacanth; sodium starch glycolate; bentonite; a naturalsponge; a surfactant; a resin such as a cation-exchange resin; citruspulp; sodium lauryl sulfate; sodium lauryl sulfate in combinationstarch; and the like.

In some embodiments, the dispersing agents suitable for the aqueoussuspensions and dispersions described herein are known in the art andinclude, for example, hydrophilic polymers, electrolytes, Tween® 60 or80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®),and the carbohydrate-based dispersing agents such as, for example,hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC,HPC-SL, and HPC-L), hydroxypropyl methylcellulose and hydroxypropylmethylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, and HPMCK100M), carboxymethylcellulose sodium, methylcellulose,hydroxyethylcellulose, hydroxypropylmethyl-cellulose phthalate,hydroxypropylmethyl-cellulose acetate stearate, noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), polyvinylpyrrolidone/vinyl acetate copolymer (Plasdone®,e.g., S-630), 4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethyleneoxide and formaldehyde (also known as tyloxapol), poloxamers (e.g.,Pluronics F68®, F88®, and F108®, which are block copolymers of ethyleneoxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, alsoknown as Poloxamine 908®, which is a tetrafunctional block copolymerderived from sequential addition of propylene oxide and ethylene oxideto ethylenediamine (BASF Corporation, Parsippany, N.J.)). In otherembodiments, the dispersing agent is selected from a group notcomprising one of the following agents: hydrophilic polymers;electrolytes; Tween® 60 or 80; PEG; polyvinylpyrrolidone (PVP);hydroxypropylcellulose and hydroxypropyl cellulose ethers (e.g., HPC,HPC-SL, and HPC-L); hydroxypropyl methylcellulose and hydroxypropylmethylcellulose ethers (e.g. HPMC K100, HPMC K4M, HPMC K15M, HPMC K100M,and Pharmacoat® USP 2910 (Shin-Etsu)); carboxymethylcellulose sodium;methylcellulose; hydroxyethylcellulose; hydroxypropylmethyl-cellulosephthalate; hydroxypropylmethyl-cellulose acetate stearate;non-crystalline cellulose; magnesium aluminum silicate; triethanolamine;polyvinyl alcohol (PVA); 4-(1,1,3,3-tetramethylbutyl)-phenol polymerwith ethylene oxide and formaldehyde; poloxamers (e.g., Pluronics F68®,F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); or poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®).

Wetting agents suitable for the aqueous suspensions and dispersionsdescribed herein are known in the art and include, but are not limitedto, cetyl alcohol, glycerol monostearate, polyoxyethylene sorbitan fattyacid esters (e.g., the commercially available Tweens® such as e.g.,Tween 20® and Tween 80® (ICI Specialty Chemicals)), and polyethyleneglycols (e.g., Carbowaxs 3350® and 1450®, and Carbopol 934® (UnionCarbide)), oleic acid, glyceryl monostearate, sorbitan monooleate,sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitanmonooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodiumlauryl sulfate, sodium docusate, triacetin, vitamin E TPGS, sodiumtaurocholate, simethicone, phosphotidylcholine and the like.

Suitable preservatives for the aqueous suspensions or dispersionsdescribed herein include, for example, potassium sorbate, parabens(e.g., methylparaben and propylparaben), benzoic acid and its salts,other esters of parahydroxybenzoic acid such as butylparaben, alcoholssuch as ethyl alcohol or benzyl alcohol, phenolic compounds such asphenol, or quaternary compounds such as benzalkonium chloride.Preservatives, as used herein, are incorporated into the dosage form ata concentration sufficient to inhibit microbial growth.

Suitable viscosity enhancing agents for the aqueous suspensions ordispersions described herein include, but are not limited to, methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropylcellulose, hydroxypropylmethyl cellulose, Plasdon® S-630, carbomer,polyvinyl alcohol, alginates, acacia, chitosans and combinationsthereof. The concentration of the viscosity enhancing agent will dependupon the agent selected and the viscosity desired.

Examples of sweetening agents suitable for the aqueous suspensions ordispersions described herein include, for example, acacia syrup,acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream,berry, black currant, butterscotch, calcium citrate, camphor, caramel,cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citruspunch, citrus cream, cotton candy, cocoa, cola, cool cherry, coolcitrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose,fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup,grape, grapefruit, honey, isomalt, lemon, lime, lemon cream,monoammonium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple,marshmallow, menthol, mint cream, mixed berry, neohesperidine DC,neotame, orange, pear, peach, peppermint, peppermint cream, Prosweet®Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol,spearmint, spearmint cream, strawberry, strawberry cream, stevia,sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfamepotassium, mannitol, talin, sucralose, sorbitol, swiss cream, tagatose,tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wildcherry, wintergreen, xylitol, or any combination of these flavoringingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange,cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint,menthol-eucalyptus, orange-cream, vanilla-mint, and mixtures thereof. Inone embodiment, the aqueous liquid dispersion can comprise a sweeteningagent or flavoring agent in a concentration ranging from about 0.001% toabout 1.0% the volume of the aqueous dispersion. In another embodiment,the aqueous liquid dispersion can comprise a sweetening agent orflavoring agent in a concentration ranging from about 0.005% to about0.5% the volume of the aqueous dispersion. In yet another embodiment,the aqueous liquid dispersion can comprise a sweetening agent orflavoring agent in a concentration ranging from about 0.01% to about1.0% the volume of the aqueous dispersion.

In addition to the additives listed above, the liquid formulations canalso include inert diluents commonly used in the art, such as water orother solvents, solubilizing agents, and emulsifiers. Exemplaryemulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodiumdoccusate, cholesterol, cholesterol esters, taurocholic acid,phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

In some embodiments, the pharmaceutical formulations described hereincan be self-emulsifying drug delivery systems (SEDDS). Emulsions aredispersions of one immiscible phase in another, usually in the form ofdroplets. Generally, emulsions are created by vigorous mechanicaldispersion. SEDDS, as opposed to emulsions or microemulsions,spontaneously form emulsions when added to an excess of water withoutany external mechanical dispersion or agitation. An advantage of SEDDSis that only gentle mixing is required to distribute the dropletsthroughout the solution. Additionally, water or the aqueous phase can beadded just prior to administration, which ensures stability of anunstable or hydrophobic active ingredient. Thus, the SEDDS provides aneffective delivery system for oral and parenteral delivery ofhydrophobic active ingredients. SEDDS may provide improvements in thebioavailability of hydrophobic active ingredients. Methods of producingself-emulsifying dosage forms are known in the art and include, but arenot limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and6,960,563, each of which is specifically incorporated by reference.

It is to be appreciated that there is overlap between the above-listedadditives used in the aqueous dispersions or suspensions describedherein, since a given additive is often classified differently bydifferent practitioners in the field, or is commonly used for any ofseveral different functions. Thus, the above-listed additives should betaken as merely exemplary, and not limiting, of the types of additivesthat can be included in formulations described herein. The amounts ofsuch additives can be readily determined by one skilled in the art,according to the particular properties desired.

Intranasal Formulations

Intranasal formulations are known in the art and are described in, forexample, U.S. Pat. Nos. 4,476,116, 5,116,817 and 6,391,452, each ofwhich is specifically incorporated by reference. Formulations thatinclude ibrutinib and/or an additional therapeutic agent, which areprepared according to these and other techniques well-known in the artare prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, fluorocarbons, and/or other solubilizing ordispersing agents known in the art. See, for example, Ansel, H. C. etal., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed.(1995). Preferably these compositions and formulations are prepared withsuitable nontoxic pharmaceutically acceptable ingredients. Theseingredients are known to those skilled in the preparation of nasaldosage forms and some of these can be found in REMINGTON: THE SCIENCEAND PRACTICE OF PHARMACY, 21st edition, 2005, a standard reference inthe field. The choice of suitable carriers is highly dependent upon theexact nature of the nasal dosage form desired, e.g., solutions,suspensions, ointments, or gels. Nasal dosage forms generally containlarge amounts of water in addition to the active ingredient. Minoramounts of other ingredients such as pH adjusters, emulsifiers ordispersing agents, preservatives, surfactants, gelling agents, orbuffering and other stabilizing and solubilizing agents may also bepresent. The nasal dosage form should be isotonic with nasal secretions.

For administration by inhalation described herein may be in a form as anaerosol, a mist or a powder. Pharmaceutical compositions describedherein are conveniently delivered in the form of an aerosol spraypresentation from pressurized packs or a nebulizer, with the use of asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, such as, by way of example only, gelatin foruse in an inhaler or insufflator may be formulated containing a powdermix of the compound described herein and a suitable powder base such aslactose or starch.

Buccal Formulations

Buccal formulations may be administered using a variety of formulationsknown in the art. For example, such formulations include, but are notlimited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and5,739,136, each of which is specifically incorporated by reference. Inaddition, the buccal dosage forms described herein can further include abioerodible (hydrolysable) polymeric carrier that also serves to adherethe dosage form to the buccal mucosa. The buccal dosage form isfabricated so as to erode gradually over a predetermined time period,wherein the delivery is provided essentially throughout. Buccal drugdelivery, as will be appreciated by those skilled in the art, avoids thedisadvantages encountered with oral drug administration, e.g., slowabsorption, degradation of the active agent by fluids present in thegastrointestinal tract and/or first-pass inactivation in the liver. Withregard to the bioerodible (hydrolysable) polymeric carrier, it will beappreciated that virtually any such carrier can be used, so long as thedesired drug release profile is not compromised, and the carrier iscompatible with ibrutinib and/or an additional therapeutic agent, andany other components that may be present in the buccal dosage unit.Generally, the polymeric carrier comprises hydrophilic (water-solubleand water-swellable) polymers that adhere to the wet surface of thebuccal mucosa. Examples of polymeric carriers useful herein includeacrylic acid polymers and co, e.g., those known as “carbomers”(Carbopol®, which may be obtained from B.F. Goodrich, is one suchpolymer). Other components may also be incorporated into the buccaldosage forms described herein include, but are not limited to,disintegrants, diluents, binders, lubricants, flavoring, colorants,preservatives, and the like. For buccal or sublingual administration,the compositions may take the form of tablets, lozenges, or gelsformulated in a conventional manner.

Transdermal Formulations

Transdermal formulations described herein may be administered using avariety of devices which have been described in the art. For example,such devices include, but are not limited to, U.S. Pat. Nos. 3,598,122,3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636,3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084,4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303,5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and6,946,144, each of which is specifically incorporated by reference inits entirety.

The transdermal dosage forms described herein may incorporate certainpharmaceutically acceptable excipients which are conventional in theart. In one embodiments, the transdermal formulations described hereininclude at least three components: (1) a formulation of a compound ofibrutinib and an additional therapeutic agent; (2) a penetrationenhancer; and (3) an aqueous adjuvant. In addition, transdermalformulations can include additional components such as, but not limitedto, gelling agents, creams and ointment bases, and the like. In someembodiments, the transdermal formulation can further include a woven ornon-woven backing material to enhance absorption and prevent the removalof the transdermal formulation from the skin. In other embodiments, thetransdermal formulations described herein can maintain a saturated orsupersaturated state to promote diffusion into the skin.

Formulations suitable for transdermal administration of compoundsdescribed herein may employ transdermal delivery devices and transdermaldelivery patches and can be lipophilic emulsions or buffered, aqueoussolutions, dissolved and/or dispersed in a polymer or an adhesive. Suchpatches may be constructed for continuous, pulsatile, or on demanddelivery of pharmaceutical agents. Still further, transdermal deliveryof the compounds described herein can be accomplished by means ofiontophoretic patches and the like. Additionally, transdermal patchescan provide controlled delivery of ibrutinib and an additionaltherapeutic agent. The rate of absorption can be slowed by usingrate-controlling membranes or by trapping the compound within a polymermatrix or gel. Conversely, absorption enhancers can be used to increaseabsorption. An absorption enhancer or carrier can include absorbablepharmaceutically acceptable solvents to assist passage through the skin.For example, transdermal devices are in the form of a bandage comprisinga backing member, a reservoir containing the compound optionally withcarriers, optionally a rate controlling barrier to deliver the compoundto the skin of the host at a controlled and predetermined rate over aprolonged period of time, and means to secure the device to the skin.

Injectable Formulations

Formulations that include a compound of ibrutinib and/or an additionaltherapeutic agent, suitable for intramuscular, subcutaneous, orintravenous injection may include physiologically acceptable sterileaqueous or non-aqueous solutions, dispersions, suspensions or emulsions,and sterile powders for reconstitution into sterile injectable solutionsor dispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection may also contain additives such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the growth ofmicroorganisms can be ensured by various antibacterial and antifungalagents, such as parabens, chlorobutanol, phenol, sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, such as aluminum monostearate and gelatin.

For intravenous injections, compounds described herein may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. For transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art. For other parenteral injections, appropriateformulations may include aqueous or nonaqueous solutions, preferablywith physiologically compatible buffers or excipients. Such excipientsare generally known in the art.

Parenteral injections may involve bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The pharmaceutical composition described herein may be ina form suitable for parenteral injection as a sterile suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Pharmaceutical formulations for parenteral administrationinclude aqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

Other Formulations

In certain embodiments, delivery systems for pharmaceutical compoundsmay be employed, such as, for example, liposomes and emulsions. Incertain embodiments, compositions provided herein can also include anmucoadhesive polymer, selected from among, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, the compounds described herein may be administeredtopically and can be formulated into a variety of topicallyadministrable compositions, such as solutions, suspensions, lotions,gels, pastes, medicated sticks, balms, creams or ointments. Suchpharmaceutical compounds can contain solubilizers, stabilizers, tonicityenhancing agents, buffers and preservatives.

The compounds described herein may also be formulated in rectalcompositions such as enemas, rectal gels, rectal foams, rectal aerosols,suppositories, jelly suppositories, or retention enemas, containingconventional suppository bases such as cocoa butter or other glycerides,as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and thelike. In suppository forms of the compositions, a low-melting wax suchas, but not limited to, a mixture of fatty acid glycerides, optionallyin combination with cocoa butter is first melted.

Diagnostic Methods

Disclosed herein are methods of using biomarkers for stratification ofpatients, for monitoring the progression of a treatment, or foroptimization of a therapeutic regimen. In some embodiments, thebiomarkers are evaluated based on the presence or absence ofmodifications or mutations in the biomarkers, or by expression level. Insome embodiments, the biomarkers include heregulin. In some embodiments,the treatment or therapeutic regimen comprises a combination of an ACKinhibitor and an additional therapeutic agent. In some embodiments, theadditional therapeutic agent is an anti-HER2 therapeutic agent, apan-ErbB inhibitor, or an anti-VEGF therapeutic agent. In someembodiments, the anti-HER2 therapeutic agent is selected fromtrastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. Insome embodiments, the pan-ErbB inhibitor is selected from afatinib,neratinib, and dacomitinib. In some embodiments, the anti-VEGFtherapeutic agent is selected from bevacizumab, ranibizumab, lapatinib,sunitinib, sorafenib, axitinib, and pazopanib. In some embodiments, theadditional therapeutic agent is selected from temsirolimus, paclitaxel,ASLAN001 (also, ARRY-543, ASLAN Pharmaceuticals), vorinostat,doxorubicin, cyclophosphamide, cisplatin, docetaxel, and dasatinib. Insome embodiments, the additional therapeutic agent is selected fromtrastuzumab and docetaxel; pertuzumab and docetaxel; doxorubicin,cyclophosphamide, and paclitaxal; or doxorubicin, cyclophosphamide, and5-FU. In some embodiments, the ACK inhibitor is a BTK inhibitor. In someembodiments, the ACK inhibitor is selected from among ibrutinib(PCI-32765), PCI-45292, PCI-45466, AVL-101/CC-101 (AvilaTherapeutics/Celgene Corporation), AVL-263/CC-263 (AvilaTherapeutics/Celgene Corporation), AVL-292/CC-292 (AvilaTherapeutics/Celgene Corporation), AVL-291/CC-291 (AvilaTherapeutics/Celgene Corporation), CNX 774 (Avila Therapeutics),BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb),CGI-1746 (CGI Pharma/Gilead Sciences), CGI-560 (CGI Pharma/GileadSciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also, CTK4I7891,HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5,AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (OnoPharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486(Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited) andLFM-A13. In some embodiments, the ACK inhibitor compound is(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one(i.e. PCI-32765/ibrutinib). In some embodiments, HRG induces resistancein HER2-amplified tumor. In some embodiments, ibrutinib sensitizesHRG-induced resistant breast cancer cells. In some embodiments, elevatedlevels of ibrutinib sensitize HRG-induced resistant breast cancer cells.In some embodiments, the breast cancer cells include cells from theBT-474 and MDA-MB-453 cell lines.

In some embodiments, the expression level of heregulin is 0.5-fold,1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold,5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold,9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 75-fold, 100-fold,200-fold, 500-fold, 1000-fold, or more compared to the reference levelof heregulin. In some embodiments, the expression level of heregulin is0.5-fold, 1-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold,4.5-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold,8.5-fold, 9-fold, 9.5-fold, 10-fold, 15-fold, 20-fold, 50-fold, 75-fold,100-fold, 200-fold, 500-fold, 1000-fold, or less compared to thereference level of heregulin.

In some embodiments, the reference level is the expression level ofheregulin in an individual who does not have HER2 amplified tumor. Insome embodiments, the reference level is the expression level ofheregulin in an individual prior to treatment with a combination of anACK inhibitor and an additional therapeutic agent.

Methods for determining the expression level or the presence ofbiomarkers such as heregulin are well known in the art, for example, byELISA, radioimmunoassay (RIA), electrochemiluminescence (ECL), Westernblot, multiplexing technologies, or other similar methods. Cell surfaceexpression of biomarkers are measured, for example, by flow cytometry,immunohistochemistry, Western Blot, immunoprecipitation, magnetic beadselection, and quantification of cells expressing either of these cellsurface markers. Biomarker RNA expression levels could be measured byRT-PCR, Qt-PCR, microarray, Northern blot, or other similartechnologies.

As disclosed herein, determining the expression or presence of thebiomarker of interest at the protein or nucleotide level is accomplishedusing any detection method known to those of skill in the art. By“detecting expression” or “detecting the level of is intendeddetermining the expression level or presence of a biomarker protein orgene in the biological sample. Thus, “detecting expression” encompassesinstances where a biomarker is determined not to be expressed, not to bedetectably expressed, expressed at a low level, expressed at a normallevel, or overexpressed.

In certain aspects of the method provided herein, the tumor samples areisolated, detected or measured. In certain embodiments, the tumorsamples are isolated, detected or measured using immunophenotypingtechniques. In other embodiments, the tumor samples are isolated,detected or measured using fluorescence activated cell sorting (FACS)techniques.

In certain aspects, the expression or presence of these variousbiomarkers and any clinically useful prognostic markers in a biologicalsample are detected at the protein or nucleic acid level, using, forexample, immunohistochemistry techniques or nucleic acid-basedtechniques such as in situ hybridization and RT-PCR. In one embodiments,the expression or presence of one or more biomarkers is carried out by ameans for nucleic acid amplification, a means for nucleic acidsequencing, a means utilizing a nucleic acid microarray (DNA and RNA),or a means for in situ hybridization using specifically labeled probes.

In other embodiments, determining the expression or presence of one ormore biomarkers is carried out through gel electrophoresis. In oneembodiment, the determination is carried out through transfer to amembrane and hybridization with a specific probe.

In other embodiments, the determining the expression or presence of oneor more biomarkers carried out by a diagnostic imaging technique.

In still other embodiments, the determining the expression or presenceof one or more biomarkers carried out by a detectable solid substrate.In one embodiment, the detectable solid substrate is paramagneticnanoparticles functionalized with antibodies.

In some embodiments, expression level of a biomarker protein of interestin a biological sample is detected by means of a binding protein capableof interacting specifically with that biomarker protein or abiologically active variant thereof. In some embodiments, labeledantibodies, binding portions thereof, or other binding partners areused. The word “label” when used herein refers to a detectable compoundor composition that is conjugated directly or indirectly to the antibodyso as to generate a “labeled” antibody. In some embodiments, the labelis detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, catalyzes chemicalalteration of a substrate compound or composition that is detectable.

The antibodies for detection of a biomarker protein are eithermonoclonal or polyclonal in origin, or are synthetically orrecombinantly produced. The amount of complexed protein, for example,the amount of biomarker protein associated with the binding protein, forexample, an antibody that specifically binds to the biomarker protein,is determined using standard protein detection methodologies known tothose of skill in the art. A detailed review of immunological assaydesign, theory and protocols are found in numerous texts in the art(see, for example, Ausubel et al., eds. (1995) Current Protocols inMolecular Biology) (Greene Publishing and Wiley-Interscience, NY));Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley& Sons, Inc., New York, N.Y.).

The choice of marker used to label the antibodies will vary dependingupon the application. However, the choice of the marker is readilydeterminable to one skilled in the art. These labeled antibodies areused in immunoassays as well as in histological applications to detectthe presence of any biomarker or protein of interest. The labeledantibodies are either polyclonal or monoclonal. Further, the antibodiesfor use in detecting a protein of interest are labeled with aradioactive atom, an enzyme, a chromophoric or fluorescent moiety, or acolorimetric tag as described elsewhere herein. The choice of tagginglabel also will depend on the detection limitations desired. Enzymeassays (ELISAs) typically allow detection of a colored product formed byinteraction of the enzyme-tagged complex with an enzyme substrate.Radionuclides that serve as detectable labels include, for example,I-131, I-123, I-125, Y-90, Re-188, Re-186, At-211, Cu-67, Bi-212, andPd-109. Examples of enzymes that serve as detectable labels include, butare not limited to, horseradish peroxidase, alkaline phosphatase,beta-galactosidase, and glucose-6-phosphate dehydrogenase. Chromophoricmoieties include, but are not limited to, fluorescein and rhodamine. Theantibodies are conjugated to these labels by methods known in the art.For example, enzymes and chromophoric molecules are conjugated to theantibodies by means of coupling agents, such as dialdehydes,carbodiimides, dimaleimides, and the like. Alternatively, conjugationoccurs through a ligand-receptor pair. Examples of suitableligand-receptor pairs are biotin-avidin or biotin-streptavidin, andantibody-antigen.

In certain embodiments, expression or presence of the biomarkersdisclosed herein is determined by radioimmunoassays or enzyme-linkedimmunoassays (ELISAs), competitive binding enzyme-linked immunoassays,dot blot (see, for example, Promega Protocols and Applications Guide,Promega Corporation (1991), Western blot (see, for example, Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual, Vol. 3, Chapter 18(Cold Spring Harbor Laboratory Press, Plainview, N.Y.), chromatographysuch as high performance liquid chromatography (HPLC), or other assaysknown in the art. Thus, the detection assays involve steps such as, butnot limited to, immunoblotting, immunodiffusion, immunoelectrophoresis,or immunoprecipitation.

In some embodiments, the expression or presence of the biomarkersdescribed herein are also determined at the nucleic acid level. Nucleicacid-based techniques for assessing expression are well known in the artand include, for example, determining the level of biomarker mRNA in abiological sample. Many expression detection methods use isolated RNA.Any RNA isolation technique that does not select against the isolationof mRNA is utilized for the purification of RNA (see, e.g., Ausubel etal., ed. (1987-1999) Current Protocols in Molecular Biology (John Wiley& Sons, New York). Additionally, large numbers of tissue samples arereadily processed using techniques well known to those of skill in theart, such as, for example, the single-step RNA isolation processdisclosed in U.S. Pat. No. 4,843,155.

Thus, in some embodiments, the detection of a biomarker or other proteinof interest is assayed at the nucleic acid level using nucleic acidprobes. The term “nucleic acid probe” refers to any molecule that iscapable of selectively binding to a specifically intended target nucleicacid molecule, for example, a nucleotide transcript. Probes aresynthesized by one of skill in the art, or derived from appropriatebiological preparations. Probes are specifically designed to be labeled,for example, with a radioactive label, a fluorescent label, an enzyme, achemiluminescent tag, a colorimetric tag, or other labels or tags thatare discussed above or that are known in the art. Examples of moleculesthat are utilized as probes include, but are not limited to, RNA andDNA.

For example, isolated mRNA are used in hybridization or amplificationassays that include, but are not limited to, Southern or Northernanalyses, polymerase chain reaction analyses and probe arrays. Onemethod for the detection of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that hybridize to the mRNAencoded by the gene being detected. The nucleic acid probe comprises of,for example, a full-length cDNA, or a portion thereof, such as anoligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to an mRNA or genomic DNA encoding a biomarker, biomarkerdescribed herein above. Hybridization of an mRNA with the probeindicates that the biomarker or other target protein of interest isbeing expressed.

In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in a gene chip array. A skilled artisan readilyadapts known mRNA detection methods for use in detecting the level ofmRNA encoding the biomarkers or other proteins of interest.

An alternative method for determining the level of an mRNA of interestin a sample involves the process of nucleic acid amplification, e.g., byRT-PCR (see, for example, U.S. Pat. No. 4,683,202), ligase chainreaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189 193),self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwohet al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi et al. (1988) Bio/Technology 6:1197), rolling circlereplication (U.S. Pat. No. 5,854,033) or any other nucleic acidamplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers. In particular aspects of the invention, biomarker expression isassessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan System).

Expression levels of an RNA of interest are monitored using a membraneblot (such as used in hybridization analysis such as Northern, dot, andthe like), or microwells, sample tubes, gels, beads or fibers (or anysolid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934. The detectionof expression also comprises using nucleic acid probes in solution.

In one embodiment of the invention, microarrays are used to determineexpression or presence of one or more biomarkers. Microarrays areparticularly well suited for this purpose because of the reproducibilitybetween different experiments. DNA microarrays provide one method forthe simultaneous measurement of the expression levels of large numbersof genes. Each array consists of a reproducible pattern of captureprobes attached to a solid support. Labeled RNA or DNA is hybridized tocomplementary probes on the array and then detected by laser scanningHybridization intensities for each probe on the array are determined andconverted to a quantitative value representing relative gene expressionlevels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135,6,033,860, and 6,344,316. High-density oligonucleotide arrays areparticularly useful for determining the gene expression profile for alarge number of RNA's in a sample.

Techniques for the synthesis of these arrays using mechanical synthesismethods are described in, e.g., U.S. Pat. No. 5,384,261. In someembodiments, an array is fabricated on a surface of virtually any shapeor even a multiplicity of surfaces. In some embodiments, an array is aplanar array surface. In some embodiments, arrays include peptides ornucleic acids on beads, gels, polymeric surfaces, fibers such as fiberoptics, glass or any other appropriate substrate, see U.S. Pat. Nos.5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. In someembodiments, arrays are packaged in such a manner as to allow fordiagnostics or other manipulation of an all-inclusive device.

In some embodiments, the sample for use in the methods is obtained fromcells of solid tumor (e.g. breast cancer cell lines). In someembodiments, the cell lines include BT-474, SK-BR3, MDA-MB-453 andUACC-893. In some embodiments, the cell lines include Mino and DoHH2cell lines.

In some embodiments, the sample is any tissue or fluid samples from apatient. Samples include, but are not limited to, blood, lymph, urine,gynecological fluids, biopsies, and smears. Bodily fluids useful for themethods disclosed herein include blood, urine, saliva, nipple aspirates,or any other bodily secretion or derivative thereof. In someembodiments, blood includes whole blood, plasma, serum, or anyderivative of blood. In some embodiments, the body sample comprisesbreast cells, for example breast tissue from a biopsy, or a breast tumortissue sample.

In some embodiments, body samples are obtained from a patient by avariety of techniques including, for example, by scraping or swabbing anarea, by using a needle to aspirate bodily fluids, or by removing atissue sample (i.e., biopsy). Methods for collecting various bodysamples are well known in the art. In some embodiments, a breast tissuesample is obtained by, for example, fine needle aspiration biopsy, coreneedle biopsy, or excisional biopsy. Fixative and staining solutions maybe applied to the cells or tissues for preserving the specimen and forfacilitating examination. Body samples, particularly breast tissuesamples, may be transferred to a glass slide for viewing undermagnification.

Kits/Articles of Manufacture

Described herein are kits for treating HER2 amplified breast cancer inan individual in need thereof comprising administering to the individuala composition comprising a therapeutically-effective amount of an ACKinhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib). Further described herein are kits for treating HER2amplified cancer in an individual in need thereof comprisingadministering to the individual a composition comprising atherapeutically-effective amount of an ACK inhibitor compound (e.g., aBTK inhibitor, such as for example ibrutinib).

For use in the therapeutic applications described herein, kits andarticles of manufacture are also described herein. In some embodiments,such kits include a carrier, package, or container that iscompartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) including one of theseparate elements to be used in a method described herein. Suitablecontainers include, for example, bottles, vials, syringes, and testtubes. The containers can be formed from a variety of materials such asglass or plastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.A wide array of formulations of the compounds and compositions providedherein are contemplated as are a variety of treatments for any disorderthat benefit by inhibition of Btk, or in which Btk is a mediator orcontributor to the symptoms or cause.

The container(s) optionally have a sterile access port (for example thecontainer is an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). Such kits optionallycomprising a compound with an identifying description or label orinstructions relating to its use in the methods described herein.

A kit will typically include one or more additional containers, eachwith one or more of various materials (such as reagents, optionally inconcentrated form, and/or devices) desirable from a commercial and userstandpoint for use of a compound described herein. Non-limiting examplesof such materials include, but not limited to, buffers, diluents,filters, needles, syringes; carrier, package, container, vial and/ortube labels listing contents and/or instructions for use, and packageinserts with instructions for use. A set of instructions will alsotypically be included.

In some embodiments, a label is on or associated with the container. Alabel can be on a container when letters, numbers or other charactersforming the label are attached, molded or etched into the containeritself; a label can be associated with a container when it is presentwithin a receptacle or carrier that also holds the container, e.g., as apackage insert. A label can be used to indicate that the contents are tobe used for a specific therapeutic application. The label can alsoindicate directions for use of the contents, such as in the methodsdescribed herein.

In certain embodiments, a pharmaceutical composition comprising the ACKinhibitor compound (e.g., a BTK inhibitor, such as for exampleibrutinib) is presented in a pack or dispenser device which can containone or more unit dosage forms. The pack can for example contain metal orplastic foil, such as a blister pack. The pack or dispenser device canbe accompanied by instructions for administration. The pack or dispensercan also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, can be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions containing a compound providedherein formulated in a compatible pharmaceutical carrier can also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

EXAMPLES

The following specific and non-limiting examples are to be construed asmerely illustrative, and do not limit the present disclosure in any waywhatsoever.

Example 1 Assays and Reagents

Cells and Reagents:

Breast cancer lines (BT-474, SK-BR3, MDA-MB-453 and UACC-893) wereobtained from ATCC and cultured as indicated from the source. Antibodieswere obtained from Cell Signaling and Santa Cruz Biotechnologies.Western blotting reagents were obtained from Life Technology(Invitrogen). Antibody to Btk was obtained from BD Biosciences.

BT-474 and SK-BR-3 cells (ATCC) were cultured in DMEM and McCoy 5Amedia, respectively, supplemented with 10% fetal calf serum. Antibodiesto EGFR, pEGFR (Y1086), HER2, pHER2 (Y1248), HER3, pHER3 (Y1289), Akt,pAkt, MEK, pMEK, and pBtk (Y223) were from Cell Signaling Technology.Antibodies to HER4, pHER4 (Y1056), ERK, pERK, and α-tubulin were fromSanta Cruz Biotechnology, Inc. Antibody to BTK was from BD Biosciences.

Kinase Activity Assay:

The LabChip platform was used for enzyme activity assay performed byNanosyn, Inc. The recombinant enzymes were used at 5 nM with substrateat 1 μM.

Cell Proliferation Assay with Alamar Blue:

Cells were plated at 10,000 cells/well in 100 μL. After 3 daysincubation at 37° C. in a 5% CO₂ incubator, alamar blue (Invitrogen1:10) was added into each well and incubated for another 2.5-3 hours.The plate was read at Excitation/Emission wavelengths of 545/590 nm.

Cell Growth Inhibition Wash Out Assay:

Cells were plated at 350,000 cells/plate for BT-474, and 300,000cells/plate for SK-BR-3 cells in 10 cm plate. Cells were treated withibrutinib for 1 h. After wash-out and fresh media added, the culturecontinued for another 6 days. Cell number in each plate was counted bycell counter after trypsinization.

Cell Cycle Analysis:

Cells were plated at 350,000 cells/plate for BT-474, and 300,000cells/plate for SK-BR-3 cells in 10 cm plate. Cells were treated withibrutinib for 1 h. After washing twice with media, the culture wascontinued for 24 h. Cells were fixed in 70% ethanol, and stained withPI/RNase. The data were acquired with BD FACSCalibur, and the cell cycleanalysis was performed using FlowJo software.

Immunoblotting:

Cells were washed once with cold PBS and lysed in 1× sample buffer(Invitrogen). The whole cell lysate was boiled, sonicated, and thenloaded onto a 4-15% gradient SDS-PAGE gel. The proteins were transferredto PVDF membrane. After probing with antibodies, the detection wasconducted using an Odyssey spectrometer (Li-Cor). Paired mouse andrabbit antibodies were used to probe the total and correspondingphosphorylated proteins.

Tumorsphere Culture:

BT-474 cells were treated with ibrutinib for 1 h at 120,000 cells in lmLmedia, in triplicate. The culture was conducted in a 6-well ultra-lowadherent plate using MammoCult media as suggested by manufacturer (StemCell Technologies). For treatment, cells were trypsinized and the singlecell suspension were treated with ibrutinib for 1 h in triplicate. Afterwashing twice, the cells were resuspended in 6 mL complete MammoCultmedia, and cultured for 7 days. For tumorsphere counting, concentratethe spheres into 1.5 mL media, and transfer into a 96-well plate. Thenumber of tumorspheres was counted using a Nikon phase-contrastmicroscope for the spheres larger than 5 mm, using a ruler installed inthe eyepiece. The size of tumorspheres was measured from photos taken inthe microscope with 4× objective. 5 pictures were taken for each well,and the size of tumorspheres in computer monitor was measured.

Aldefluor Assay:

BT-474 cells were treated with ibrutinib for 1 h, washed twice, and theculture continued for 3 days, or were cultured continuously in thepresence of 0.1 μM ibrutinib for 3 days. The assay was conductedaccording to directions provided by the manufacturer (StemCellTechnologies). Cells were incubated with the Aldefluor reagent for35 minutes prior to flow cytometry.

Western Blotting:

Whole cell lysates in 1× sample buffer (Invitrogen) were electrophoresedon a 4%-12% Bis-Tris gel. After transferring the proteins onto a PVDFmembrane, the blot was probed by antibodies, and signal was detectedusing an Odyssey imager (LI-COR Biosciences). Paired mouse and rabbitantibodies were used to probe the total and corresponding phosphorylatedproteins.

Apoptosis Assay:

Cells were stained with annexin-V/PI or PI/RNase, and apoptotic cellswere quantitated using a FACSCalibur flow cytometer (Becton Dickinson).The numbers of annexin-V positive cells or subGO cells were calculated.

Results

Inhibition of ErbB Kinases by Ibrutinib

ErbB family of kinases share high degrees of homology in their kinasedomain with Btk around a conserved cysteine residue, which can bondirreversibly to ibrutinib (FIG. 1). The ability of ibrutinib to inhibitthe various kinases illustrated in FIG. 1 was assayed. As shown in thefigure, ibrutinib was able to inhibit EGFR (ErbB1), ErB2 and Erb4 inaddition to BTK and other members of the TEC kinase family (e.g. BMX,TEC, TXK, ITK, JAK3 and BLK). In a separate experiment, it was furtherobserved that kinase inhibition was dialysis resistant (data not shown),which supports covalent inhibition.

Growth Inhibition of HER-2 Positive Breast Cancer Cells

Breast cancer lines (BT-474, SK-BR3, MDA-MB-453 and UACC-893) which areHER2 amplified were cultured in the presence of ibrutinib, AVL-292 andPCI-45468. After 3 days continuous exposure to ibrutinib, it wasobserved that the HER-2 positive breast cancer cells are sensitive tothe growth inhibitory effect of ibrutinib as assay by alamar blue assay(see e.g., FIGS. 2A-2D). After 1 hour exposure to ibrutinib followed bywash out and culturing for 6 days, it was observed that the growthinhibitory effect was sustained and was dose dependent (see, e.g., FIGS.3A and 3B). Further, one hour treatment with ibrutinib has a lastinginhibitory effect even after day 6 on HER signaling pathway (see, e.g.FIG. 33). Cell cycle analysis showed that cells with 1 h exposure toibrutinib had G1 arrest as measured at 24 h (see, e.g., FIGS. 4A and4B). For BT-474 cells, the difference was statistically significant(p<0.05) between the control and ibrutinib treated groups. For SK-BR-3cells, the difference was statistically significant (p<0.05 or p<0.01)between the control and ibrutinib treated groups, and among differentdose groups.

Ibrutinib induced apoptosis in BT-474 cells (see, e.g., FIGS. 5A-C andFIG. 17). One hour of drug treatment caused a low level of apoptosis 6days after the exposure (FIG. 5A), while continuous treatment (3-day)resulted in dramatic apoptosis which was caspase-dependent Q-VD-OPH(FIGS. 5B and 5C, and FIG. 17). Apoptosis was measured as percentage ofcells in sub G0 or with annexin-V positivity. Ibrutinib also inhibitedtumorsphere growth of BT-474 cells in culture without affectingtumorsphere number (see, e.g. FIGS. 6A-B) and inhibited solid tumorgrowth in a mouse MDA-MB-453 xenograft (see e.g. FIG. 7).

The expression of total and phosphorylated forms of EGFR, HER2, HER4,MEK, ERK, and AKT was examined in various cancer cells lines in theabsence or presence of ibrutinib (0.1 μM) after 1 hour of treatment.Breast cancer lines that were sensitive to ibrutinib are alsoHER2/4-amplified (see, e.g., FIGS. 8 and 9). In a washout experiment,where cells were treated with ibrutinib for various times and thencultured for an additional 2 hours, inhibition of ErbB kinases anddown-stream signaling pathways persisted after ibrutinib washout, andwas time and concentration dependent (see, e.g., FIG. 10).

The effect of ibrutinib on progenitor cell marker expression in BT-474breast cancer cells was analyzed by Aldefluor assay, which identifiesprogenitor cells based on expression of aldehyde dehydrogenase (ALDH).Diethylaminobenzaldehyde (DEAB) inhibits aldehyde dehydrogenase activityand was used as a control in the assay. It was observed that one hour orcontinuous treatment with ibrutinib decreased putative “stem-like”subpopulation of breast cancer cells (see, e.g., FIG. 11 and FIG. 18).For example, decreased aldehyde dehydrogenase activity detected by theAldefluor assay was noted after short exposures to high concentrationsor continuous exposure to low concentration of ibrutinib. Thus, shortexposure to ibrutinib causes a reduction of growth potential forputative “stem-like” cancer cells.

Using a fluorescence-based assay, the inhibition of the breast cancercell line MDA-MB-453 by ibrutinib or gefitinib (Iressa) was determined.Gefitinib is a reversible inhibitor that has higher EGFR inhibitoryactivity but lower HER2/4 inhibitory activity compared to ibrutinib. Itwas observed that ibrutinib was more potent than gefitinib in inhibitingMDA-MB-453 cell growth (see e.g., FIG. 12). Ibrutinib also exhibitedsimilar growth inhibitory activity in SK-BR3 cells and MDA-MB-453 breastcancer cells and as compared to lapatinib, neratinib and dacomitinib(see, e.g., FIGS. 13A and 13B). Separate experiments confirmed ibrutinibinhibitory activity in BT-474, SK-BR3 and MDA-MB-453 breast cancer cellsas compared to kinase inhibitors gefitinib, lapatinib, neratinib anddacomitinib (see, e.g., FIGS. 14A-C). Ibrutinib was also a more potentinhibitor of cell growth compared to AVL-292 in BT-474, SK-BR3 andMDA-MB-453 breast cancer cells (see, e.g., FIGS. 15A-C). In addition, ascompared to AVL-292, only ibrutinib inhibited HER2 and HER4 activationas assessed by phosphorylation of downstream targets, such as AKT (see,e.g., FIG. 16).

Example 2 HER2 Amplified Breast Cancer

Study Type: Interventional

Study Design:

Allocation: Non-Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Outcome Measures

To estimate the efficacy [overall response rate (ORR)=complete response(CR) and partial response (PR)](phase II)

To determine the expression level of HER2 and p95-HER2 in the metastatictissue samples and correlate this with response to the ibrutinib inHER2-amplified tumors. [Time Frame: once at the time of biopsy][Designated as safety issue: No]

Eligibility

Ages Eligible for Study: 18 Years and older

Genders Eligible for Study: Both

Accepts Healthy Volunteers: No

Inclusion Criteria:

Phase I HER2-Amplified Cohort

HER2 overexpression and/or amplification as determined byimmunohistochemistry (3+) or FISH (≧2.0)

Previously received trastuzumab as part of a regimen in the adjuvant ormetastatic setting with evidence of progression. Washout period fortrastuzumab of 14 days.

May have previously received lapatinib as part of a regimen in theadjuvant or metastatic setting with evidence of progression of disease.Washout period for lapatinib of 14 days.

Radiographic progression of disease while on treatment with trastuzumabor lapatinib as defined by RECIST 1.1 criteria.

No restriction on prior chemotherapy regimens for advanced stagedisease. No restriction for prior hormonal therapy. No concurrent use ofendocrine therapy is permitted.

Phase II HER2-Amplified Cohort

HER2 overexpression and/or amplification as determined byimmunohistochemistry (3+) or FISH (≧2.0).

Previously received trastuzumab as part of a regimen in the adjuvant ormetastatic setting with evidence of progression. Washout period fortrastuzumab of 14 days.

May have previously received lapatinib as part of a regimen in theadjuvant or metastatic setting with evidence of progression of disease.Washout period for lapatinib of 14 days.

Radiographic progression of disease while on treatment with trastuzumabas defined by RECIST 1.1 criteria.

Prior Therapy Inclusion:

No more than four prior chemotherapy regimens allowed for advanced stagedisease. No restriction for prior hormonal therapy. No concurrent use ofendocrine therapy is permitted.

Inclusion Criteria for all Subjects

Patients with a diagnosis of invasive adenocarcinoma of the breastconfirmed by histology or cytology at MSKCC.

Metastatic disease that is or has been pathologically documented.

At least one measurable metastatic lesion according to RECIST 1.1criteria. Ascites, pleural effusions, and bone metastases are notconsidered measurable. Minimum indicator lesion size ≧10 mm by helicalCT or ≧20 mm by conventional techniques.

Pathological nodes must be ≧15 mm by the short axis to be consideredmeasurable.

Age ≧18, as no dosing or adverse event data are currently available onthe use of neratinib or temsirolimus in patients <18 years of age,children are excluded from this study.

Able and willing to consent for biopsy of metastatic breast cancer priorto treatment. Consent to preservation of frozen and fixed samples oftumor cores for evaluation.

Consent to Evaluation of Primary Tumor Biopsy Specimen.

Patients must be willing to discontinue sex hormonal therapy, e.g.,birth control pills, hormonal replacement therapy, prior to enrollment.Women of childbearing potential must consent to effective contraceptionwhile on treatment and for a period thereafter.

Negative serum HCG pregnancy test for premenopausal women ofreproductive capacity and for women less than 12 months after menopause.

Asymptomatic, central nervous system metastases are permitted ifpatients remain clinically stable after discontinuation of steroids andanticonvulsants for 3 months.

Eastern Cooperative Oncology Group (ECOG) performance status score of≦2.

Patients must have normal organ and marrow function: AST/ALT ≦2.5×institutional upper limit of normal except for patients with livermetastases. For patients with liver metastases, AST/ALT/Alkalinephosphatase ≦5.0× institutional upper limit of normal. Total bilirubinwithin institutional limits except for patients with liver metastases.For patients with liver metastases, total bilirubin ≦1.5× institutionalupper limit of normal. Creatinine clearance within normal limits or ≧60mL/min, PT and PTT≦1.5× institutional upper limit of normal except forpatients on Coumadin or low molecular weight heparin, leukocytes≧3,000/μl, absolute neutrophil count ≧1,000/μl, and platelets ≧75,000/μl

Able to swallow and retain oral medication.

Exclusion Criteria:

Potential subjects will be excluded from enrollment into this study ifthey meet any of the following criteria:

Patients receiving any concurrent anticancer therapy or investigationalagents with the intention of treating breast cancer.

History of allergic reactions attributed to compounds of similarchemical or biologic composition to neratinib or temsirolimus.

Unable to consent to biopsy of metastatic disease or for whom a biopsywould be medically unsafe.

Women who are pregnant or breast feeding.

Life expectancy <3 months.

Completion of previous chemotherapy regimen <3 weeks prior to the startof study treatment. Prior hormonal therapy must be discontinued prior totreatment start. Biologic therapy with bevacizumab for the treatment ofmetastatic disease must be discontinued ≧3 weeks from the start ofprotocol treatment.

Concurrent radiotherapy is not permitted for disease progression ontreatment on protocol, but might be allowed for pre-existing non-targetlesions with approval from the principal investigator of the trial.

Concurrent medical conditions which may increase the risk of toxicity,including ongoing or active infection, history of significant bleedingdisorder unrelated to cancer (congenital bleeding disorders, acquiredbleeding disorders within one year), HIV-positive or active hepatitis.

History of clinically significant or uncontrolled cardiac disease,including congestive heart failure, angina, myocardial infarction,arrhythmia, and left ventricular ejection fraction less than 50%measured by a multigated blood pool imaging of the heart (MUGA scan) oran echocardiogram (ECHO).

QTc interval >0.47 seconds.

Patients with GI tract disease resulting in an inability to take oralmedication, malabsorption syndrome, a requirement for IV alimentation,prior surgical procedures affecting absorption, or uncontrolledinflammatory GI disease.

History of an invasive second primary malignancy diagnosed within theprevious 3 years, except for stage I endometrial or cervical carcinomaor prostate carcinoma treated surgically, and non-melanoma skin cancer.

History of uncontrolled seizures, central nervous system disorders orpsychiatric disability judged by the investigator to be clinicallysignificant, precluding informed consent, or interfering with complianceof oral drug intake.

Unwillingness to give written informed consent, unwillingness toparticipate, or inability to comply with the protocol for the durationof the study. Willingness and ability to comply with scheduled visits,treatment plan, laboratory tests and other study procedures arenecessary to participation in this clinical trial.

Example 3 HER2 Amplified Gastric Cancer

Study Type: Interventional

Study Design:

Allocation: Non-Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Outcome Measures

The percentage of patients demonstrating clear evidence of inhibition ofreceptor auto-phosphorylation in HER-2 amplified patients on Day 30.

Objective Response Rate

Eligibility

Ages Eligible for Study: 21 Years and older

Genders Eligible for Study: Both

Accepts Healthy Volunteers: No

Inclusion Criteria:

Male or female patients 21 years of age or older at the time writteninformed consent is obtained.

Patients with histologically confirmed adenocarcinoma of the stomach,gastro-oesophageal junction or distal oesophagus with inoperablelocally-advanced metastatic disease.

Patients with tumours with gene-amplification of HER-2 by standard FISH.

Patient has received 1 or more prior chemotherapy for the treatment ofadenocarcinoma of the stomach, gastro-oesophageal junction or distaloesophagus with metastatic disease.

Patients with prior partial gastrectomy if they can take oralmedications, and can undergo gastroendoscopic biopsies and meet allother inclusion/exclusion criteria.

Patients with measurable and non-measurable disease per modified RECISTguidelines. All scans and x-rays used to document measurable ornon-measurable disease must be done within a 28-day period prior toenrollment.

Patient with Eastern Cooperative Oncology Group (ECOG) performancestatus of 0 or 1 (within 14 days prior to enrolment).

Patient with adequate organ and hematological function as evidenced bythe following laboratory studies within 14 days prior to enrollment:

Hematological function, as follows: Absolute neutrophil count(ANC)≧1.5×109/L; Platelet count ≧75×109/L; Hemoglobin ≧9 g/dL;

Coagulation functions, as follows: Partial thromboplastin time (PTT) oractivated partial thromboplastin time (aPTT)≦1.5× upper limits of normal(ULN) per institutional laboratory normal range; Internationalnormalized ratio (INR)≦1.5

Renal functions, as follows: Serum creatinine ≦1.5×ULN; Urea ≦1.5×ULN

Hepatic function, as follows: Total bilirubin ≦1.5×ULN; Serum glutamicoxaloacetic transaminase (SGOT)/aspartate transaminase (AST) and serumglutamic pyruvic transaminase (SGPT)/alanine transaminase (ALT)≦2.5×ULN(≦5×ULN if liver metastases are present)

Exclusion Criteria:

Patients unable to swallow oral medications

Patients with persistent gastric outlet obstruction, complete dysphagiaor feeding jejunostomy.

Patients who underwent radiotherapy to the gastric remnant ≦14 daysprior to enrolment. Patients must have recovered from allradiotherapy-related toxicities.

Patients with total gastrectomy.

Patients who have uncontrolled, clinically significant symptomaticcardiovascular diseases within 6 months prior to enrolment, includingmyocardial infarction, unstable angina, grade 2 or greater peripheralvascular disease, cerebrovascular accident, transient ischemic attack,congestive heart failure or arrhythmias not controlled by outpatientmedication.

Pregnant (i.e., positive beta-human chorionic gonadotropin test) or isbreast-feeding women.

Example 4 HER2 Amplified Lung Cancer

Study Type: Interventional

Study Design:

Allocation: Non-Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Outcome Measures

Progression-Free Survival (PFS); Progression Free Survival rate (PFS) at4 months [Cohort A] PFS is defined as the interval from enrollment todate of objective progression or death due to any cause.

Duration of Response (DR) per cohort

Overall Survival (OS) per cohort

Patient Reported Outcomes of health related quality of life anddisease/treatment-related symptoms as measured by the EuropeanOrganization for Research and Treatment of Cancer Quality of LifeQuestionnaire (EORTC QLQ-C30), and Lung Cancer module (LC13)

Eligibility

Ages Eligible for Study: 21 Years and older

Genders Eligible for Study: Both

Accepts Healthy Volunteers: No

Inclusion Criteria:

Advanced adenocarcinoma of lung, measurable disease

Patients with tumours with gene-amplification of HER-2 by standard FISHregardless of smoking status

ECOG (Eastern Cooperative Oncology Group) 0-1.

Cohort A: No prior systemic therapy

Cohort B: patients with HER2 amplified NSCLC; may have had prior therapy

Exclusion Criteria:

Active brain metastases

Prior systemic therapy for advanced disease in Cohort A only. Cohort Bcan have had any number of prior lines of systemic therapy.

Example 5

Cell lines BT-474, Mino, and DoHH2 were obtained from ATCC and culturedas indicated from the source.

Kinase Activity Assay:

Kinase activity assay was performed using the LabChip platform byNanosyn, Inc. using 5 nM of recombinant enzymes (EGFR, HER2, and HER4)and various concentrations of the inhibitors (e.g. ibrutinib, afatinib,neratinib, dacomitinib, lapatinib, and gefitinib).

For DoHH2 cells, the following protocol was used. DoHH2 cells (1million/mL) were treated with inhibitors for 1 h, followed with 30 mintreatment of PCI-33380 (2 uM). Cells were then pelleted and resuspendedin 24 uL PBS with protease inhibitors. Next, the cells were freeze-thaw4 times. 24 uL supernatant of the cell lysate was mixed with 8 uL 4×Invitrogen sample buffer. About 15 uL of the cell lysate mixture wasloaded onto each well of an electrophoresis gel.

The inhibition of the signaling pathway by ibrutinib was compared withafatinib (see, e.g. FIG. 19). Both ibrutinib and afatinib showed greaterinhibitor, i.e. higher IC50, toward EGFR than for HER2 and HER4 (see,e.g. FIG. 19B). Dacomitinib, a pan-ErbB inhibitor, was less effective ininhibiting the EGFR and downstream targets (see, e.g. FIG. 20).Ibrutinib was a potent inhibitor of the MEK and Akt signaling pathwaythan of the EGFR and HER2 signaling pathway (see, e.g. FIG. 21).Furthermore, a higher concentration of ibrutinib was able to overcomeresistance induced by heregulin (see, e.g. FIG. 22). In comparison withadditional inhibitors described herein, only ibrutinib showed selectiveinhibitory effect on Btk signaling pathways in B cells (e.g. Mino cells)(see, e.g. FIG. 23). Further, ibrutinib selectively bound to Btkcovalently, while the remaining inhibitors, neratinib, lapatinib, anddacomitinib, did not (see, e.g. FIG. 24).

Example 6 Covalent Binding of Ibrutinib to ErbB Kinases

Cell lines BT474 and SK-BR-3 were obtained from ATCC and cultured asindicated from the source.

BTK Dialysis Assay:

100 nM and 10 nM compound was pre-incubated with 5 nM BTK enzyme for 1 hin a buffer comprising 100 mM HEPES pH7.5, 0.1% BSA, 5 mM MgCl2, 1 mMDTT, 0.01% Triton X-100 and was dialyzed at +4° C. against the samebuffer for a total time of 24 h (3 changes of the dialysis buffer,nominal cumulative dialysis factor: 8,000,000). Control samples includedDMSO+5 nM BTK dialyzed in the identical manner. Un-dialyzed samples withcompound were assembled and pre-incubated for 1 hr. Following dialysis,the BTK activity was measured in real-time format in the presence of 250uM ATP and 1 uM substrate peptide. Initial velocity was determined inthe samples.

LCK Dialysis Assay:

100 nM and 10 nM compound was pre-incubated with 5 nM LCK enzyme for 1 hin a buffer comprising 100 mM HEPES pH7.5, 0.1% BSA, 5 mM MgCl2, 1 mMDTT, 0.01% Triton X-100 and was dialyzed at +4° C. against the samebuffer for a total time of 24 h (3 changes of the dialysis buffer,nominal cumulative dialysis factor: 8,000,000). Control samplesincluded: DMSO+5 nM LCK dialyzed in the identical manner. Un-dialyzedsamples with compound were assembled and pre-incubated for 1 hr.Following dialysis, the LCK activity was measured in real-time format inthe presence of 50 uM ATP and 1 uM substrate peptide. Initial velocitywas determined in the samples.

EGFR Dialysis Assay:

100 nM and 10 nM compound was pre-incubated with 5 nM EGFR enzyme for 1h in a buffer comprising 100 mM HEPES pH7.5, 0.1% BSA, 5 mM MgCl2, 1 mMDTT, 0.01% Triton X-100 and dialyzed at +4° C. against the same bufferfor a total time of 24 h (3 changes of the dialysis buffer, nominalcumulative dialysis factor: 8,000,000). Control samples included: DMSO+5nM EGFR dialyzed in the identical manner. Un-dialyzed samples withcompound were assembled and pre-incubated for 1 hr. Following dialysis,the EGFR activity was measured in real-time format in the presence of250 uM ATP and 1 uM substrate peptide. Initial velocity was determinedin the samples.

HER4 Dialysis Assay:

100 nM and 10 nM compound was pre-incubated with 5 nM HER4 enzyme for 1h in a buffer comprising 100 mM HEPES pH7.5, 0.1% BSA, 5 mM MgCl2, 1 mMDTT, 0.01% Triton X-100 and dialyzed at +4° C. against the same bufferfor a total time of 24 h (3 changes of the dialysis buffer, nominalcumulative dialysis factor: 8,000,000). Control samples included: DMSO+5nM HER4 dialyzed in the identical manner. Un-dialyzed samples withcompound were assembled and pre-incubated for 1 hr. Following dialysis,the HER4 activity was measured in real-time format in the presence of250 uM ATP and 1 uM substrate peptide. Initial velocity of wasdetermined in the samples.

Recombinant enzymes BTK, EGFR, and HER4 were dialyzed against either 10nM or 100 nM concentration of ibrutinib. Ibrutinib (PCI-32765)demonstrated irreversible inhibition of BTK (see, e.g. FIG. 25). In thepresence of LCK, ibrutinib demonstrated reversible inhibition (see, e.g.FIG. 26). Lymphocyte-specific protein tyrosine kinase (LCK) served as anegative control. LCK lacks the conserved cysteine residue in its kinasedomain, and its activity was recovered following dialysis after 1 hpre-exposure to ibrutinib. In the presence of EGFR and HER4, ibrutinibshowed both irreversible inhibition to both recombinant enzymes (see,e.g. FIG. 27 for EGFR inhibition and FIG. 28 for HER4 inhibition). Theenzymes in DMSO (control) were dialyzed as well.

Example 7 Time Dependent Inhibition by Ibrutinib (PCI-32765) Thru RapidDilution

Ibrutinib (PCI-32765) was tested against two recombinant kinases, Btkand HER2. Ibrutinib was tested at 0.1 μM concentration. Ibrutinib orDMSO (control) was pre-incubated with either Btk or HER2 for 5 min, 15min, 30 min, 60 min and 90 min (the pre-incubation samples wereassembled in reverse order: 90 min, 60 min, 30 min, 15 min, and 5 min).The Ibrutinib/kinase complexes were rapidly diluted (dilution factor500×) into assay buffer with 500 uM ATP and 2 uM substrate peptide. Realtime enzyme activity assay was performed in the diluted samples. Thetime-dependent irreversibility of the inhibitor was determined by thedifference between the treated enzymes diluted in the presence ofcompound and without compound. The initial velocity of the reaction wasplotted against pre-incubation time to determine the apparent Kobs ofinhibition. FIG. 29 illustrates the time-dependent inhibition of BTK byibrutinib after rapid dilution. FIG. 30 illustrates the time-dependentinhibition of HER2 by ibrutinib after rapid dilution.

Determining Kinetics of Inhibition by Ibrutinib (PCI-32765)

Ibrutinib (PCI-32765) was tested in BTK and HER2 assays. Compound wasserially pre-diluted in DMSO. The serial dilutions were transferred into35 mL of 1× assay buffer supplemented with 4 mM substrate peptide. Thereactions were initiated by addition of 35 uL of 2× enzyme (0.5 nM BTKor 1.0 nM HER2 in 1× assay buffer supplemented with ATP.

The final BTK assay composition comprised 100 mM HEPES pH7.5, 0.1% BSA,0.01% Triton X-100, 1 mM DTT, 5 mM MgCl₂, 1000 μM ATP, 0.25 nM BTK(Millipore), and 2 μM peptide. The temperature was at 25° C. DMSOconcentration was at 0.05%. The peptide sequence wasFAM-GEEPLYWSFPAKKK-NH₂.

The final HER2 assay composition comprised 100 mM HEPES pH7.5, 0.1% BSA,0.01% Triton X-100, 1 mM DTT, 5 mM MnCl₂, 1000 μM ATP, 0.5 nM HER2(BPS), and 2 μM peptide. The temperature was at 25° C. DMSOconcentration was at 0.05%. The peptide sequence wasFAM-GEEPLYWSFPAKKK-NH₂.

The reaction progress curves were obtained for a total time of ˜4 hrsusing climate controlled Caliper LabChip® instrument. The obtainedcurves were fitted using XLfit4 software using best fitting equation.For example: for time dependent inhibition, the equation:[P]=Vs*t+((Vo−Vs)/Kobs)*(1−exp(−Kobs*t)) was used. In the equation: Viis the initial velocity of the reaction and the Vs is the steady statevelocity in the presence of inhibitor.

For time dependent inhibitors: the obtained Kobs values were plottedagainst compound concentration using either hyperbolic fit. From theplots, the K2 (or Kinact), K-2, Ki and Kinact/Ki values were determined.

FIG. 31 illustrates the kinetics of BTK inhibition by ibrutinib(PCI-32765). FIG. 32 illustrates the kinetics of HER2 inhibition byibrutinib (PCI-32765).

The examples and embodiments described herein are for illustrativepurposes only and various modifications or changes suggested to personsskilled in the art are to be included within the spirit and purview ofthis application and scope of the appended claims.

What is claimed is:
 1. A method for treating a refractory HER2-amplifiedbreast cancer in an individual in need thereof comprising administeringto an individual in need thereof a composition comprising(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one


2. The method of claim 1, wherein the refractory HER2-amplified breastcancer is metastatic.
 3. The method of claim 1, wherein the refractoryHER2-amplified breast cancer is refractory to a treatment oftrastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, or MM-111. 4.The method of claim 1, wherein the refractory HER2-amplified breastcancer has a HER2:CEP17 ratio of 2.2-4.0 or >4.0.
 5. The method of claim1, further comprising co-administering an additional therapeutic agent.6. The method of claim 5, wherein the additional therapeutic agent is ananti-HER2 therapeutic agent, a pan-ErbB inhibitor, or an anti-VEGFtherapeutic agent.
 7. The method of claim 6, wherein the anti-HER2therapeutic agent is selected from the group consisting of: trastuzumab,trastuzumab emtansine, pertuzumab, lapatinib, and MM-111.
 8. The methodof claim 6, wherein the pan-ErbB inhibitor is selected from the groupconsisting of: afatinib, neratinib, and dacomitinib.
 9. The method ofclaim 6, wherein the anti-VEGF therapeutic agent is selected from thegroup consisting of: bevacizumab, ranibizumab, lapatinib, sunitinib,sorafenib, axitinib, and pazopanib.
 10. The method of claim 5, whereinthe additional therapeutic agent is selected from the group consistingof: temsirolimus; paclitaxel; varlitinib; vorinostat; doxorubicin;cyclophosphamide; cisplatin; docetaxel; dasatinib; trastuzumab anddocetaxel; pertuzumab and docetaxel; doxorubicin, cyclophosphamide andpaclitaxal; and doxorubicin, cyclophosphamide and fluorouracil.
 11. Amethod for treating a refractory HER2 amplified cancer in an individualin need thereof comprising administering to an individual in needthereof a composition comprising(R)-1-(3-(4-amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one


12. The method of claim 11, wherein the refractory HER2-amplified canceris selected from the group consisting of: breast, colon, endometrial,cervical, urothelial, lung (including, non-small cell lung cancer),ovarian, gastric, gastroesophageal junction (GEJ), head and neck,biliary tract, prostate, and pancreatic cancer.
 13. The method of claim11, wherein the refractory HER2-amplified cancer is metastatic.
 14. Themethod of claim 11, wherein the refractory HER2-amplified cancer has aHER2:CEP17 ratio of 2.2-4.0 or >4.0.
 15. The method of claim 11, whereinthe refractory HER2-amplified cancer is refractory to a treatment oftrastuzumab, trastuzumab emtansine, pertuzumab, lapatinib, or MM-111.16. The method of claim 11, further comprising co-administering anadditional therapeutic agent.
 17. The method of claim 16, wherein theadditional therapeutic agent is an anti-HER2 therapeutic agent, apan-ErbB inhibitor, or an anti-VEGF therapeutic agent.
 18. The method ofclaim 17, wherein the anti-HER2 therapeutic agent is selected from thegroup consisting of: trastuzumab, trastuzumab emtansine, pertuzumab,lapatinib, and MM-111.
 19. The method of claim 17, wherein the pan-ErbBinhibitor is selected from the group consisting of: afatinib, neratinib,and dacomitinib.
 20. The method of claim 17, wherein the anti-VEGFtherapeutic agent is selected from the group consisting of: bevacizumab,ranibizumab, lapatinib, sunitinib, sorafenib, axitinib, and pazopanib.21. The method of claim 16, wherein the additional therapeutic agent isselected from the group consisting of: temsirolimus; paclitaxel;varlitinib; vorinostat; doxorubicin; cyclophosphamide; cisplatin;docetaxel; dasatinib; trastuzumab and docetaxel; pertuzumab anddocetaxel; doxorubicin, cyclophosphamide and paclitaxal; anddoxorubicin, cyclophosphamide and fluorouracil.